U.S. patent application number 10/104949 was filed with the patent office on 2003-01-23 for modulation of molecular interaction sites on rna and other biomolecules.
Invention is credited to Crooke, Stanley T., Ecker, David J., Griffey, Richard, Hofstadler, Steven, Mohan, Venkatraman, Sampath, Ranga, Swayze, Eric.
Application Number | 20030017483 10/104949 |
Document ID | / |
Family ID | 22131813 |
Filed Date | 2003-01-23 |
United States Patent
Application |
20030017483 |
Kind Code |
A1 |
Ecker, David J. ; et
al. |
January 23, 2003 |
Modulation of molecular interaction sites on RNA and other
biomolecules
Abstract
Methods for the identification of compounds which modulate,
either inhibit or stimulate, biomolecules are provided. Nucleic
acids, especially RNAs are preferred substrates for such
modulation. The present methods are particularly powerful in that
they provide novel combinations of techniques which give rise to
compounds, usually "small" organic compounds, which are highly
potent modulators of RNA and other biomolecular activity. In
accordance with preferred aspects of the invention, very large
numbers of compounds may be tested essentially simultaneously to
determine whether they are likely to interact with a molecular
interaction site and modulate the activity of the biomolecule.
Pharmaceuticals, veterinary drugs, agricultural chemicals,
industrial chemicals, research chemicals and many other beneficial
compounds may be identified in accordance with embodiments of this
invention.
Inventors: |
Ecker, David J.; (Encinitas,
CA) ; Griffey, Richard; (Vista, CA) ; Crooke,
Stanley T.; (Carlsbad, CA) ; Sampath, Ranga;
(San Diego, CA) ; Swayze, Eric; (Carlsbad, CA)
; Mohan, Venkatraman; (Carlsbad, CA) ; Hofstadler,
Steven; (Oceanside, CA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE, 46TH FLOOR
1650 MARKET STREET
PHILADELPHIA
PA
19103
US
|
Family ID: |
22131813 |
Appl. No.: |
10/104949 |
Filed: |
March 22, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10104949 |
Mar 22, 2002 |
|
|
|
09076404 |
May 12, 1998 |
|
|
|
Current U.S.
Class: |
435/6.14 ;
702/20; 703/11 |
Current CPC
Class: |
C12N 15/113 20130101;
C12N 15/1048 20130101; C40B 40/00 20130101 |
Class at
Publication: |
435/6 ; 702/20;
703/11 |
International
Class: |
C12Q 001/68; G06G
007/48; G06G 007/58; G06F 019/00 |
Claims
What is claimed is:
1. A method of identifying a compound which modulates activity of a
target RNA comprising identifying at least one molecular
interaction site on said target RNA generating in silico a virtual
library of compounds predicted or calculated to interact with said
molecular interaction site; and comparing three dimensional
representations of said target RNA with members of the virtual
library of compounds to generate a hierarchy of said compounds
ranked in accordance with their respective ability to form physical
interactions with said molecular interaction site.
2. The method of claim 1 further comprising synthesizing the highly
ranked members of said hierarchy of compounds.
3. The method of claim 2 further comprising testing said highly
ranked members to determine their ability to interact with said
molecular interaction site.
4. The method of claim 2 further comprising contacting the target
RNA with at least one of said highly ranked members to provide a
complex between the RNA and the member or members; ionizing said
complex; fragmenting the ionized complex; and determining whether
highly ranked members binds to the molecular interaction site of
said RNA.
5. The method of claim 4 further comprising determining the
strength of binding of a highly ranked member in comparison to the
binding strength of other highly ranked members.
6. A method of identifying a compound which modulates activity of a
target biolmolecule comprising identifying at least one molecular
interaction site on said target biomolecule; generating in silico a
virtual library of compounds predicted or calculated to interact
with said molecular interaction site; and comparing three
dimensional representations of said target biomolecule with members
of the virtual library of compounds to generate a hierarchy of said
compounds ranked in accordance with their respective ability to
form physical interactions with said molecular interaction
site.
7. The method of claim 6 further comprising synthesizing the highly
ranked members of said hierarchy of compounds.
8. The method of claim 7 further comprising testing said highly
ranked members to determine their ability to interact with said
molecular interaction site.
9. The method of claim 7 further comprising contacting the target
biomolecule with at least one of said highly ranked members to
provide a complex between the RNA and the member or members;
ionizing said complex; fragmenting the ionized complex; and
determining whether highly ranked members binds to the molecular
interaction site of said biomolecule.
10. The method of claim 9 further comprising determining the
strength of binding of a highly ranked member in comparison to the
binding strength of other highly ranked members.
11. A compound identified in accordance with claim 1.
12. A method of modulating the action of an RNA comprising
contacting said RNA with a compound identified in accordance with
claim 1.
13 A pharmaceutical, agricultural chemical or industrial chemical
comprising a compound identified in accordance with claim 1.
14. A compound identified in accordance with claim 6.
15. A method of modulating the action of a biomolecule comprising
contacting said RNA with a compound identified in accordance with
claim 6.
16. A pharmaceutical, agricultural chemical or industrial chemical
comprising a compound identified in accordance with claim 6.
17. A method of identifying a compound which modulates activity of
a target RNA comprising generating in silico a virtual library of
compounds predicted or calculated to interact with said RNA;
comparing three dimensional representations of said target RNA with
members of the virtual library of compounds to generate a hierarchy
of said compounds ranked in accordance with their respective
ability to form physical interactions with said molecular
interaction site; and synthesizing the highly ranked members of
said hierarchy of compounds.
18. The method of claim 17 further comprising testing said highly
ranked members to determine their ability to interact with said
molecular interaction site.
19. The method of claim 18 further comprising contacting the target
RNA with at least one of said highly ranked members to provide a
complex between the RNA and the member or members; ionizing said
complex; fragmenting the ionized complex; and determining whether
highly ranked members binds to the molecular interaction site of
said RNA.
20. The method of claim 19 further comprising determining the
strength of binding of a highly ranked member in comparison to the
binding strength of other highly ranked members.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the identification of
compounds which modulate, either inhibit or stimulate,
biomolecules. Nucleic acids, especially RNA are preferred
substrates for such modulation and all such substrates are
denominated "targets" for such action. The present methods are
particularly powerful in that they provide novel combinations of
techniques which give rise to compounds, usually "small" organic
compounds, which are highly potent modulators of RNA and other
biomolecular activity. Very large numbers of compounds may be
tested in silico to determine whether they are likely to interact
with a molecular interaction site and, hence, modulate the activity
of the biomolecule. Pharmaceuticals, veterinary drugs, agricultural
chemicals, industrial chemicals, research chemicals and many other
beneficial compounds may be identified in accordance with
embodiments of this invention.
BACKGROUND OF THE INVENTION
[0002] Recent advances in genomics, molecular biology, and
structural biology have highlighted how RNA molecules participate
in or control many of the events required to express proteins in
cells. Rather than function as simple intermediaries, RNA molecules
actively regulate their own transcription from DNA, splice and edit
mRNA molecules and tRNA molecules, synthesize peptide bonds in the
ribosome, catalyze the migration of nascent proteins to the cell
membrane, and provide fine control over the rate of translation of
messages. RNA molecules can adopt a variety of unique structural
motifs, which provide the framework required to perform these
functions.
[0003] "Small" molecule therapeutics, which bind specifically to
structured RNA molecules, are organic chemical molecules which are
not polymers. "Small" molecule therapeutics include the most
powerful naturally-occurring antibiotics. For example, the
aminoglycoside and macrolide antibiotics are "small" molecules that
bind to defined regions in ribosomal RNA (rRNA) structures and
work, it is believed, by blocking conformational changes in the RNA
required for protein synthesis. Changes in the conformation of RNA
molecules have been shown to regulate rates of transcription and
translation of mRNA molecules.
[0004] An additional opportunity in targeting RNA for drug
discovery is that cells frequently create different mRNA molecules
in different tissues that can be translated into identical
proteins. Processes such as alternative splicing and alternative
polyadenylation can create transcripts that are unique or enriched
in particular tissues. This provides the opportunity to design
drugs that bind to the region of RNA unique in a desired tissue,
including tumors, and not affect protein expression in other
tissues, or affect protein expression to a lesser extent, providing
an additional level of drug specificity generally not achieved by
therapeutic targeting of proteins.
[0005] RNA molecules or groups of related RNA molecules are
believed by Applicants to have regulatory regions that are used by
the cell to control synthesis of proteins. The cell is believed to
exercise control over both the timing and the amount of protein
that is synthesized by direct, specific interactions with mRNA.
This notion is inconsistent with the impression obtained by reading
the scientific literature on gene regulation, which is highly
focused on transcription. The process of RNA maturation, transport,
intracellular localization and translation are rich in RNA
recognition sites that provide good opportunities for drug binding.
Applicants' invention is directed to finding these regions for RNA
molecules in the human genome as well as in other animal genomes
and prokaryotic genomes.
[0006] Combinatorial chemistry is a recent addition to the toolbox
of chemists and represents a field of chemistry dealing with the
synthesis of a large number of chemical entities. This is generally
achieved by condensing a small number of reagents together in all
combinations defined by a given reaction sequence. Advances in this
area of chemistry include the use of chemical software tools and
advanced computer hardware which has made it possible to consider
possibilities for synthesis in orders of magnitude greater than the
actual synthesis of the library compounds. The concept of "virtual
library" is used to indicate a collection of candidate structures
that would theoretically result from a combinatorial synthesis
involving reactions of interest and reagents to effect those
reactions. It is from this virtual library that compounds are
selected to be actually synthesized.
[0007] Project Library (MDL Information Systems, Inc., San Leandro,
Calif.) is said to be a desktop software system which supports
combinatorial research efforts. (Practical Guide to Combinatorial
Chemistry, A. W. Czarnik and S. H. DeWitt, eds., 1997, ACS,
Washington, D.C.) The software is said to include an
information-management module for the representation and search of
building blocks, individual molecules, complete combinatorial
libraries, and mixtures of molecules, and other modules for
computational support for tracking mixture and discrete-compound
libraries.
[0008] Molecular Diversity Manager (Tripos, Inc., St. Louis, Mo.)
is said to be a suite of software modules for the creation,
selection, and management of compound libraries. (Practical Guide
to Combinatorial Chemistry, A. W. Czarnik and S. H. DeWitt, eds.,
1997, ACS, Washington, D.C.) The LEGION and SELECTOR modules are
said to be useful in creating libraries and characterizing
molecules in terms of both 2-dimensional and 3-dimensional
structural fingerprints, substituent parameters, topological
indices, and physicochemical parameters.
[0009] Afferent Systems (San Francisco, Calif.) is said to offer
combinatorial library software that creates virtual molecules for a
database. It is said to do this by virtually reacting precursor
molecules and selecting those that could be actually synthesized
(Wilson, C&EN, Apr. 27, 1998, p.32).
[0010] While only Project Library and Molecular Diversity Manager
are available commercially, these products do not provide
facilities to efficiently track reagents and synthesis conditions
employed for the introduction of fragments into the desired
compounds being generated. Further, these products are unable to
track mixtures of compounds that are generated by the introduction
of multiple fragments by the use of multiple reagents. Therefore,
it is desirable to have available methods for handling mixtures of
compounds, as well as methods for the tracking of chemical
reactions or transformations utilized in the synthesis of
individual compounds and mixtures thereof.
[0011] Combinatorial chemistry is a recent addition to the toolbox
of chemists and represents a field of chemistry dealing with the
synthesis of a large number of chemical entities. This is generally
achieved by condensing a small number of reagents together in all
combinations defined by a given reaction sequence. Advances in this
area of chemistry include the use of chemical software tools and
advanced computer hardware which has made it possible to consider
possibilities for synthesis in orders of magnitude greater than the
actual synthesis of the library compounds. The concept of "virtual
library" is used to indicate a collection of candidate structures
that would theoretically result from a combinatorial synthesis
involving reactions of interest and reagents to effect those
reactions. It is from this virtual library that compounds are
selected to be actually synthesized.
[0012] Project Library (MDL Information Systems, Inc., San Leandro,
Calif.) is said to be a desktop software system which supports
combinatorial research efforts. (Practical Guide to Combinatorial
Chemistry, A. W. Czarnik and S. H. DeWitt, eds., 1997, ACS,
Washington, D.C.) The software is said to include an
information-management module for the representation and search of
building blocks, individual molecules, complete combinatorial
libraries, and mixtures of molecules, and other modules for
computational support for tracking mixture and discrete-compound
libraries.
[0013] Molecular Diversity Manager (Tripos, Inc., St. Louis, Mo.)
is said to be a suite of software modules for the creation,
selection, and management of compound libraries. (Practical Guide
to Combinatorial Chemistry, A. W. Czarnik and S. H. DeWitt, eds.,
1997, ACS, Washington, D.C.) The LEGION and SELECTOR modules are
said to be useful in creating libraries and characterizing
molecules in terms of both 2-dimensional and 3-dimensional
structural fingerprints, substituent parameters, topological
indices, and physicochemical parameters.
[0014] Afferent Systems (San Francisco, Calif.) is said to offer
combinatorial library software that creates virtual molecules for a
database. It is said to do this by virtually reacting precursor
molecules and selecting those that could be actually synthesized
(Wilson, C&EN, Apr. 27, 1998, p.32).
[0015] While only Project Library and Molecular Diversity Manager
are available commercially, these products do not provide
facilities to efficiently track the reagents employed for the
introduction of fragments into the desired compounds being
generated. Further, these products are unable to track mixtures of
compounds that are generated by the introduction of multiple
fragments by the use of multiple reagents. Therefore, it is
desirable to have available methods for handling mixtures of
compounds, as well as methods for the tracking of chemical
reactions or transformations utilized in the synthesis of
individual compounds and mixtures thereof.
[0016] The selection of compounds for synthesis and screening is a
critical step in any drug discovery process. This is particularly
true for combinatorial chemistry-based discovery strategies, where
a very much larger number of compounds can be conceived than can be
prepared in a reasonable time frame. Computational chemistry
methods have been applied to find the "best" sets of compounds for
screening. One strategy optimizes the chemical "diversity" in a
library in order to increase the likelihood of finding a hit with
biological activity in a screen against a macromolecular target of
unknown structure.
[0017] Targeting nucleic acids has been recognized as a valid
strategy for interference with biological pathways and the
treatment of disease. In this regard, both deoxyribonucleic acids
(DNA) and ribonucleic acids (RNA) have been the target of numerous
therapeutic strategies. A wide variety of "small" molecules,
oligomers and oligonucleotides have been shown to possess binding
affinity for nucleic acids. The vast majority of experience in
interfering with nucleic acid function has been via the specific
binding of ligands to a particular base, base pair, and/or primary
sequence of bases in the nucleic acid target. Some compounds have
also demonstrated a composite specificity that arises from
recognition and interactions with both the primary and secondary
structural features of the nucleic acid, such as preferential
binding to A-T base pairs in the DNA minor groove, with little or
no binding to corresponding RNA sequences.
[0018] Exploiting the knowledge of the three-dimensional structure
of biological targets is a promising strategy from a drug design
and discovery standpoint. This has been demonstrated by the design
and development of numerous drugs and drug candidates targeted to
proteins involved in various pathophysiological pathways. While
three dimensional structures of proteins have been widely
determined by techniques such as X-ray crystallography, molecular
modeling and NMR, nucleic acid targets have been difficult to
study. The literature reveals few three dimensional structures of
biologically active RNA, including a tRNA, said to have been
determined via X-ray crystallography. Quigley, et al., Nucleic
Acids Res., 1975, 2, 2329; and Moras, et al., Nature (London),
1980, 288, 669. The difficulties associated with proper
crystallization and study of nucleic acids by X-ray methods along
with the increasing number of biologically important small RNAs
have increased the need for new structure determination and drug
discovery strategies for such targets.
[0019] Many approaches to predicting RNA structure have been
discussed in the scientific literature. Essentially, these involve
sequencing and genomic analysis of nucleic acids, such as RNA, as a
first step to establish the primary sequence structure and
potential folded structures of the target. A second step entails
definition of structural constraints such as base pairing and long
range interactions among bases based on information derived from
cross-linking, biochemical and genetic structure-function studies.
This information, together with modeling and simulation software,
has allowed scientists to predict three dimensional models of RNA
and DNA. While such models may not be as powerful as X-ray crystal
structures, they have been useful in ascertaining some structural
features and structure-function relationships.
[0020] An understanding of the structural features of specific
motifs in nucleic acids, especially hairpins, loops, helices and
double helices, has been found to be useful in gaining molecular
insights. For example, a hairpin motif comprising a double helical
stem and a single-stranded loop is believed to be one of the
simplest yet most important structural element in nucleic acids.
Such hairpin structures are proposed to be nucleation sites and
serve as major building blocks for the folded three dimensional
structure of RNAs. Shen, et al., FASEB J., 1995, 9, 1023. Hairpins
are also involved in specific interactions with a variety of
proteins to regulate gene expression. Feng, et al., Nature, 1988,
334, 165, Witherell, et al., Prog. Nucleic Acids Res. Mol. Biol.,
1991, 40, 185, and Phillipe, et al., J. Mol. Biol., 1990, 211, 415.
Nucleic acid hairpin structures have therefore been widely studied
by NMR, molecular modeling techniques such as constrained molecular
dynamics and distance geometry (Cheong, et al., Nature, 1990, 346,
680 and Cain, et al., Nuc. Acids Res., 1995, 23, 2153), X-ray
crystallography (Valegard, et al., Nature, 1994, 371, 623 and
Chattopadhyaya, et al., Nature, 1988, 334, 175), and theoretical
methods (Tung, Biophysical J., 1997, 72, 876, Erie, et al.,
Biopolymers, 1993, 33, 75, and Raghunathan, et al., Biochemistry,
1991, 30, 782.
[0021] The determination of potential three dimensional structures
of nucleic acids and their attendant structural motifs affords
insights into areas such as the study of catalysis by RNA, RNA-RNA
interactions, RNA-nucleic acid interactions, RNA-protein
interactions, and the recognition of small molecules by nucleic
acids. Four general approaches to the generation of model three
dimensional structures of RNA have been demonstrated in the
literature. All of these employ sophisticated molecular modeling
and computational algorithms for the simulation of folding and
tertiary interactions within target nucleic acids, such as RNA.
Westhof and Altman (Proc. Natl. Acad. Sci., 1994, 91, 5133) have
described the generation of a three-dimensional working model of Ml
RNA, the catalytic RNA subunit of RNase P from E. coli via an
interactive computer modeling protocol. Leveraging the significant
body of work in the area of cryo-electron microscopy (cryo-EM) and
biochemical studies on ribosomal RNAs, Mueller and Brimacombe (J.
Mol. Biol., 1997, 271, 524) have constructed a three dimensional
model of E. coli 16S Ribosomal RNA. A method to model nucleic acid
hairpin motifs has been developed based on a set of reduced
coordinates for describing nucleic acid structures and a sampling
algorithm that equilibriates structures using Monte Carlo (MC)
simulations (Tung, Biophysical J., 1997, 72, 876, incorporated
herein by reference in its entirety). MC-SYM is yet another
approach to predicting the three dimensional structure of RNAs
using a constraint-satisfaction method. Major, et al., Proc. Natl.
Acad. Sci., 1993, 90, 9408. The MC-SYM program is an algorithm
based on constraint satisfaction that searches conformational space
for all models that satisfy query input constraints, and is
described in, for example, Cedergren, et al., RNA Structure And
Function, 1998, Cold Spring Harbor Lab. Press, p.37-75. Three
dimensional structures of RNA are produced by that method by the
stepwise addition of nucleotide having one or several different
conformations to a growing oligonucleotide model.
[0022] Westhof and Altman (Proc. Natl. Acad. Sci., 1994, 91, 5133)
have described the generation of a three-dimensional working model
of M1 RNA, the catalytic RNA subunit of RNase P from E. coli via an
interactive computer modeling protocol. This modeling protocol
incorporated data from chemical and enzymatic protection
experiments, phylogenetic analysis, studies of the activities of
mutants and the kinetics of reactions catalyzed by the binding of
substrate to M1 RNA. Modeling was performed for the most part as
described in the literature. Westhof, et al., in "Theoretical
Biochemistry and Molecular Biophysics," Beveridge and Lavery
(eds.), Adenine, N.Y., 1990, 399. In general, starting with the
primary sequence, of M1 RNA, the stem-loop structures and other
elements of secondary structure were created. Subsequent assembly
of these elements into a three dimensional structure using a
computer graphics station and FRODO (Jones, J. Appl. Crystallogr.,
1978, 11, 268) followed by refinement using NUCLIN-NUCLSQ afforded
a RNA model that had correct geometries, the absence of bad
contacts, and appropriate stereochemistry. The model so generated
was found to be consistent with a large body of empirical data on
M1 RNA and opens the door for hypotheses about the mechanism of
action of RNase P. However, the models generated by this method are
less well resolved that the structures determined via X-ray
crystallography.
[0023] Mueller and Brimacombe (J. Mol. Biol., 1997, 271, 524) have
constructed a three dimensional model of E. coli 16S ribosomal RNA
using a modeling program called ERNA-3D. This program generates
three dimensional structures such as A-form RNA helices and
single-strand regions via the dynamic docking of single strands to
fit electron density obtained from low resolution diffraction data.
After helical elements have been defined and positioned in the
model, the configurations of the single strand regions is adjusted,
so as to satisfy any known biochemical constraints such as
RNA-protein cross-linking and foot-printing data.
[0024] A method to model nucleic acid hairpin motifs has been
developed based on a set of reduced coordinates for describing
nucleic acid structures and a sampling algorithm that equilibriates
structures using Monte Carlo (MC) simulations. Tung, Biophysical
J., 1997, 72, 876, incorporated herein by reference. The stem
region of a nucleic acid can be adequately modeled by using a
canonical duplex formation. Using a set of reduced coordinates, an
algorithm that is capable of generating structures of single
stranded loops with a pair of fixed ends was created. This allows
efficient structural sampling of the loop in conformational space.
Combining this algorithm with a modified Metropolis Monte Carlo
algorithm afforded a structure simulation package that simplifies
the study of nucleic acid hairpin structures by computational
means.
[0025] Knowledge and mastery of the foregoing techniques is assumed
to be part of the ordinary skill in the art. There has been a
long-felt need in the art to provide methods for improved
determination of the three-dimensional structure of important
regulatory and other elements in nucleic acids, especially RNA. It
is also been greatly desired to achieve improved knowledge about
the nature of interactions between ligands and potential ligands or
nucleic acids, especially RNA. The present invention is directed
towards satisfaction of these objectives.
[0026] The process of drug discovery is changing at a fast pace
because of the rapid progress and evolution of a number of
technologies that impact this process. Drug discovery has evolved
from what was, several decades ago, essentially random screening of
natural products, into a scientific process that not only includes
the rational and combinatorial design of large numbers of synthetic
molecules as potential bioactive agents, such as ligands, agonists,
antagonists, and inhibitors, but also the identification, and
mechanistic and structural characterization of their biological
targets, which may be polypeptides, proteins, or nucleic acids.
These key areas of drug design and structural biology are of
tremendous importance to the understanding and treatment of
disease. However, significant hurdles need to be overcome when
trying to identify or develop high affinity ligands for a
particular biological target. These include the difficulty
surrounding the task of elucidating the structure of targets and
targets to which other molecules may be bound or associated, the
large numbers of compounds that need to be screened in order to
generate new leads or to optimize existing leads, the need to
dissect structural similarities and dissimilarities between these
large numbers of compounds, correlating structural features to
activity and binding affinity, and the fact that small structural
changes can lead to large effects on biological activities of
compounds.
[0027] Traditionally, drug discovery and optimization have involved
the expensive and time-consuming, and therefore slow, process of
synthesis and evaluation of single compounds bearing incremental
structural changes. When using natural products, the individual
components of extracts had to be painstakingly separated into pure
constituent compounds prior to biological evaluation. Further, all
compounds had to be carefully analyzed and characterized prior to
in vitro screening. These screens typically included evaluation of
candidate compounds for binding affinity to their target,
competition for the ligand binding site, or efficacy at the target
as determined via inhibition, cell proliferation, activation or
antagonism end points. Considering all these facets of drug design
and screening that slow the process of drug discovery, a number of
approaches to alleviate or remedy these matters, have been
implemented by those involved in discovery efforts.
[0028] One way in which the drug discovery process is being
accelerated is by the generation of large collections, libraries,
or arrays of compounds. The strategy of discovery has moved from
selection of drug leads from among compounds that are individually
synthesized and tested to the screening of large collections of
compounds. These collections may be from natural sources (Sternberg
et al., Proc. Natl. Acad. Sci. USA, 1995, 92, 1609-1613) or
generated by synthetic methods such as combinatorial chemistry
(Ecker and Crooke, Bio/Technology, 1995, 13, 351-360 and U.S. Pat.
No. 5,571,902, incorporated herein by reference). These collections
of compounds may be generated as libraries of individual,
well-characterized compounds synthesized, e.g. via high throughput,
parallel synthesis or as a mixture or a pool of up to several
hundred or even several thousand molecules synthesized by split-mix
or other combinatorial methods. Screening of such combinatorial
libraries has usually involved a binding assay to determine the
extent of ligand-receptor interaction (Chu et al., J. Am. Chem.
Soc., 1996, 118, 7827-35). Often the ligand or the target receptor
is immobilized onto a surface such as a polymer bead or plate.
Following detection of a binding event, the ligand is released and
identified. However, solid phase screening assays can be rendered
difficult by non-specific interactions.
[0029] Whether screening of combinatorial libraries is performed
via solid-phase, solution methods or otherwise, it can be a
challenge to identify those components of the library that bind to
the target in a rapid and effective manner and which, hence, are of
greatest interest. This is a process that needs to be improved to
achieve ease and effectiveness in combinatorial and other drug
discovery processes. Several approaches to facilitating the
understanding of the structure of biopolymeric and other
therapeutic targets have also been developed so as to accelerate
the process of drug discovery and development. These include the
sequencing of proteins and nucleic acids (Smith, in Protein
Sequencing Protocols, Humana Press, Totowa, N.J., 1997; Findlay and
Geisow, in Protein Sequencing: A Practical Approach, IRL Press,
Oxford, 1989; Brown, in DNA Sequencing, IRL Oxford University
Press, Oxford, 1994; Adams, Fields and Venter, in Automated DNA
Sequencing and Analysis, Academic Press, San Diego, 1994). These
also include elucidating the secondary and tertiary structures of
such biopolymers via NMR (Jefson, Ann. Rep. in Med. Chem., 1988,
23, 275; Erikson and Fesik, Ann. Rep. in Med. Chem., 1992, 27,
271-289), X-ray crystallography (Erikson and Fesik, Ann. Rep. in
Med. Chem., 1992, 27, 271-289) and the use of computer algorithms
to attempt the prediction of protein folding (Copeland, in Methods
of Protein Analysis: A Practical Guide to Laboratory Protocols,
Chapman and Hall, New York, 1994; Creighton, in Protein Folding, W.
H. Freeman and Co., 1992). Experiments such as ELISA (Kemeny and
Challacombe, in ELISA and other Solid Phase Immunoassays:
Theoretical and Practical Aspects; Wiley, New York, 1988) and
radioligand binding assays (Berson and Yalow, Clin. Chim. Acta,
1968, 22, 51-60; Chard, in "An Introduction to Radioimmunoassay and
Related Techniques," Elsevier press, Amsterdam/New York, 1982), the
use of surface-plasmon resonance (Karlsson, Michaelsson and
Mattson, J. Immunol. Methods, 1991, 145, 229; Jonsson et al.,
Biotechniques, 1991, 11, 620), and scintillation proximity assays
(Udenfriend, Gerber and Nelson, Anal. Biochem., 1987, 161, 494-500)
are being used to understand the nature of the receptor-ligand
interaction.
[0030] All of the foregoing paradigms and techniques are now
available to persons of ordinary skill in the art and their
understanding and mastery is assumed herein.
[0031] Likewise, advances have occurred in the chemical synthesis
of compounds for high-throughput biological screening.
Combinatorial chemistry, computational chemistry, and the synthesis
of large collections of mixtures of compounds or of individual
compounds have all facilitated the rapid synthesis of large numbers
of compounds for in vitro screening. Despite these advances, the
process of drug discovery and optimization entails a sequence of
difficult steps. This process can also be an expensive one because
of the costs involved at each stage and the need to screen large
numbers of individual compounds. Moreover, the structural features
of target receptors can be elusive.
[0032] One step in the identification of bioactive compounds
involves the determination of binding affinity of test compounds
for a desired biopolymeric or other receptor, such as a specific
protein or nucleic acid or combination thereof. For combinatorial
chemistry, with its ability to synthesize, or isolate from natural
sources, large numbers of compounds for in vitro biological
screening, this challenge is magnified. Since combinatorial
chemistry generates large numbers of compounds or natural products,
often isolated as mixtures, there is a need for methods which allow
rapid determination of those members of the library or mixture that
are most active or which bind with the highest affinity to a
receptor target.
[0033] From a related perspective, there are available to the drug
discovery scientist a number of tools and techniques for the
structural elucidation of biologically interesting targets, for the
determination of the strength and stoichiometry of target-ligand
interactions, and for the determination of active components of
combinatorial mixtures.
[0034] Techniques and instrumentation are available for the
sequencing of biological targets such as proteins and nucleic acids
(e.g. Smith, in Protein Sequencing Protocols, 1997 and Findlay and
Geisow, in Protein Sequencing: A Practical Approach, 1989) cited
previously. While these techniques are useful, there are some
classes and structures of biopolyiheric target that are not
susceptible to such sequencing efforts, and, in any event, greater
convenience and economy have been sought. Another drawback of
present sequencing techniques is their inability to reveal anything
more than the primary structure, or sequence, of the target.
[0035] While X-ray crystallography is a very powerful technique
that can allow for the determination of some secondary and tertiary
structure of biopolymeric targets (Erikson and Fesik, Ann. Rep. in
Med. Chem., 1992, 27, 271-289), this technique can be an expensive
procedure and very difficult to accomplish. Crystallization of
biopolymers is extremely challenging, difficult to perform at
adequate resolution, and is often considered to be as much an art
as a science. Further confounding the utility of X-ray crystal
structures in the drug discovery process is the inability of
crystallography to reveal insights into the solution-phase, and
therefore the biologically relevant, structures of the targets of
interest.
[0036] Some analysis of the nature and strength of interaction
between a ligand (agonist, antagonist, or inhibitor) and its target
can be performed by ELISA (Kemeny and Challacombe, in ELISA and
other Solid Phase Immunoassays: 1988), radioligand binding assays
(Berson and Yalow, Clin. 1968, Chard, in "An Introduction to
Radioimmunoassay and Related Techniques," 1982), surface-plasmon
resonance (Karlsson, Michaelsson and Mattson, 1991, Jonsson et al.,
Biotechniques, 1991), or scintillation proximity assays
(Udenfriend, Gerber and Nelson, Anal. Biochem., 1987), all cited
previously. The radioligand binding assays are typically useful
only when assessing the competitive binding of the unknown at the
biding site for that of the radioligand and also require the use of
radioactivity. The surface-plasmon resonance technique is more
straightforward to use, but is also quite costly. Conventional
biochemical assays of binding kinetics, and dissociation and
association constants are also helpful in elucidating the nature of
the target-ligand interactions.
[0037] When screening combinatorial mixtures of compounds, the drug
discovery scientist will conventionally identify an active pool,
deconvolute it into its individual members via resynthesis, and
identify the active members via analysis of the discrete compounds.
Current techniques and protocols for the study of combinatorial
libraries against a variety of biologically relevant targets have
many shortcomings. The tedious nature, high cost, multi-step
character, and low sensitivity of many of the above-mentioned
screening technologies are shortcomings of the currently available
tools. Further, available techniques do not always afford the most
relevant structural information--the structure of a target in
solution, for example. Instead they provide insights into target
structures that may only exist in the solid phase. Also, the need
for customized reagents and experiments for specific tasks is a
challenge for the practice of current drug discovery and screening
technologies. Current methods also fail to provide a convenient
solution to the need for deconvolution and identification of active
members of libraries without having to perform tedious re-syntheses
and re-analyses of discrete members of pools or mixtures.
[0038] Therefore, methods for the screening and identification of
complex chemical libraries especially combinatorial libraries are
greatly needed such that one or more of the structures of both the
target and ligand, the site of interaction between the target and
ligand, and the strength of the target-ligand interaction can be
determined. Further, in order to accelerate drug discovery, new
methods of screening combinatorial libraries are needed to provide
ways for the direct identification of the bioactive members from a
mixture and to allow for the screening of multiple biomolecular
targets in a single procedure. Straightforward methods that allow
selective and controlled cleavage of biopolymers, while also
analyzing the various fragments to provide structural information,
would be of significant value to those involved in biochemistry and
drug discovery and have long been desired. Also, it is preferred
that the methods not be restricted to one type of biomolecular
target, but instead be applicable to a variety of targets such as
nucleic acids, peptides, proteins and oligosaccharides.
[0039] Accordingly, it is a principal object of the invention to
identify molecular interaction sites in nucleic acids, especially
RNA. A further object of the invention is to identify secondary
structural elements in RNA which are highly likely to give rise to
significant therapeutic, regulatory, or other interactions with
"small" molecules and the like. Identification of tissue-enriched
unique structures in RNA is another objective of the present
invention.
[0040] It is another objective of the present invention to provide
improved characterization of interactions between RNA and other
nucleic acids and ligands or potential ligands therefor.
[0041] A further object of the invention is to compare molecular
interaction sites of RNA with compounds proposed for interaction
therewith.
[0042] In accordance with preferred embodiments of the present
invention, the comparison of molecular interaction sites of RNA
with compounds is achieved through comparison of numerical
representations of the three-dimensional structure of the molecular
interaction site with the three dimensional structure of the
ligands in a fashion such that such interactions can be compared as
to quality.
[0043] Another object of the present invention is the preparation
of hierarchies of ligands ranked or ordered in accordance with in
accordance with their ability to interact with molecular
interaction sites of RNA and other nucleic acid targets.
[0044] Yet another object of the present invention is the
establishment of databases of the numerical representations of
three-dimensional structures of molecular interaction sites of
nucleic acids and three-dimensional structures of libraries of
ligands. Such databases libraries provide powerful tools for the
elucidation of structure and interactions of molecular interaction
sites with potential ligands and predictions thereof.
[0045] A principal object of the present invention is to provide
novel methods for the determination of the structure of
biomolecular targets and ligands that interact with them and to
ascertain the nature and sites of such interactions.
[0046] A further object of the invention is to determine the
structural features of biomolecular targets such as peptides,
proteins, oligonucleotides, and nucleic acids such as the primary
sequence, the secondary and folded structures of biopolymers, and
higher order tertiary and quaternary structures of biomolecules
that result from intramolecular and intermolecular
interactions.
[0047] Yet another object of the invention is to determine the
site(s) and nature of interaction between a biomolecular target and
a binding ligand or ligands. The binding ligand may be a "small"
molecule, a biomolecule such as a peptide, oligonucleotide or
oligosaccharide, a natural product, or a member of a combinatorial
library.
[0048] A further object of the invention is to determine the
relative binding affinity or dissociation constant of ligands that
bind to biopolymer targets. Preferably, this gives rise to a
determination of relative binding affinities between a biopolymer
such as an RNA/DNA target and ligands e.g. members of
combinatorially synthesized libraries.
[0049] A still further object of the present invention is to
provide a general method for the screening of combinatorial
libraries comprising individual compounds or mixtures of compounds
against a biomolecular target such as a nucleic acid, so as to
determine which components of the library bind to the target.
[0050] An additional object of the present invention is to provide
methods for the determination of the molecular weight and structure
of those members of a combinatorial library that bind to a
biomolecular target.
[0051] Yet another object of the invention is to provide methods
for screening multiple targets such as nucleic acids, proteins, and
other biomolecules and oligomers simultaneously against a
combinatorial library of compounds.
[0052] A still further object of the invention is to ascertain the
specificity and affinity of compounds, especially "small" organic
molecules to bind to or interact with molecular interaction sites
of biological molecules, especially nucleic acids such as RNA. Such
molecules may be and preferably do form ranked hierarchies of
ligands and potential ligands for the molecular interaction sites,
ranked in accordance with predicted or calculated likelihood of
interaction with such sites.
[0053] Another object of the present invention is to alleviate the
problem of peak overlap in mass spectra generated from the analysis
of mixtures of screening targets and combinatorial or other
mixtures of compounds. In a preferred embodiment, the invention
provides methods to solve the problems of mass redundancy in
combinatorial or other mixtures of compounds, and also provides
methods to solve the problem of mass redundancy in the mixture of
targets being screened.
[0054] A further object of the invention is to provide methods for
determining the binding specificity of a ligand for a target in
comparison to a control. The present invention facilitates the
determination of selectivity, the identification of non-specific
effects and the elimination of non-specific ligands from further
consideration for drug discovery efforts.
SUMMARY OF THE INVENTION
[0055] The invention is directed to identification of novel drugs,
agricultural chemicals, industrial chemicals and the like which
operate through the modulation of biomolecules, especially RNAs. A
number of procedures and protocols are preferably integrated to
provide powerful drug and other biologically useful compound
identification.
[0056] Applicants' invention is directed to methods of identifying
secondary structures in eukaryotic and prokaryotic RNA molecules
termed "molecular interaction sites."Molecular interaction sites
are small, usually less than 30 nucleotides, independently folded,
functional subdomains contained within a larger RNA molecule.
Applicants' methods preferably comprise a family of integrated
processes that analyze nucleic acid, preferably RNA, sequences and
predict their structure and function. Applicants' methods
preferably comprise processes that execute subroutines in sequence,
where the results of one process are used to trigger a specific
course of action or provide numerical or other input to other
steps. Preferably, there are decision points in the processes where
the paths taken are determined by expert processes that make
decisions without detailed, real-time human intervention.
Automation of the analysis of RNA sequences provides the ability to
identify regulatory sites at the rate that RNA sequences become
available from genomic sequence databases and otherwise. The
invention can be used, for example, to identify molecular
interaction sites in connection with central nervous system (CNS)
disease, metabolic disease, pain, degenerative diseases of aging,
cancer, inflammatory disease, cardiovascular disease and many other
conditions. Applicants' invention can also be used, for example, to
identify molecular interaction sites, which are absent from
eukaryotes, particularly humans, which can serves as sites for
"small" molecule binding with concomitant modulation, either
augmenting or diminishing, of the RNA of prokaryotic organisms.
Human toxicity can, thus, be avoided in the treatment of viral,
bacterial or parasitic disease.
[0057] The present invention preferably identifies molecular
interaction sites in a target nucleic acid by comparing the
nucleotide sequence of the target nucleic acid with the nucleotide
sequences of a plurality of nucleic acids from different taxonomic
species, identifying at least one sequence region which is
effectively conserved among the plurality of nucleic acids and the
target nucleic acid, determining whether the conserved region has
secondary structure, and, for conserved regions having secondary
structure, identifying the secondary structures.
[0058] The present invention is also directed to databases relating
to molecular interaction sites, in eukaryotic and prokaryotic RNA.
The databases are obtained by comparing the nucleotide sequence of
the target nucleic acid with the nucleotide sequences of a
plurality of nucleic acids from different taxonomic species,
identifying at least one sequence region which is conserved among
the plurality of nucleic acids and the target nucleic acid,
determining whether the conserved region has secondary structure,
and for the conserved regions having secondary structure,
identifying the secondary structures, and compiling a group of such
secondary structures.
[0059] The present invention is also directed to oligonucleotides
comprising a molecular interaction site that is present in the RNA
of a selected organism and in the RNA of at least one additional
organism, wherein the molecular interaction site serves as a
binding site for at least one molecule which, when bound to the
molecular interaction site, modulates the expression of the RNA in
the selected organism.
[0060] The present invention is also directed to an oligonucleotide
comprising a molecular interaction site that is present in
prokaryotic RNA and in at least one additional prokaryotic RNA,
wherein the molecular interaction site serves as a binding site for
at least one molecule, when bound to the molecular interaction
site, modulates the expression of the prokaryotic RNA.
[0061] The present invention also concerns pharmaceutical
compositions comprising an oligonucleotide having a molecular
interaction site that is present in prokaryotic RNA and in at least
one additional prokaryotic RNA, wherein the molecular interaction
site serves as a binding site for at least one "small" molecule.
Such molecule, when bound to the molecular interaction site,
modulates the expression of the prokaryotic RNA. A pharmaceutical
carrier is also preferably included.
[0062] The present invention also provides pharmaceutical
compositions comprising an oligonucleotide comprising a molecular
interaction site that is present in the RNA of a selected organism
and in the RNA of at least one additional organism. The molecular
interaction site serves as a binding site for at least one molecule
that, when bound to the molecular interaction site, modulates the
expression of the RNA in the selected organism, and a
pharmaceutical carrier.
[0063] Ultimately, the methods of the present invention identify
the physical structures present in a target nucleic acid which are
of great importance to an organism in which the nucleic acid is
present. Such structures--called molecular interaction sites--are
capable of interacting with molecular species to modify the nature
or effect of the nucleic acid. This may be exploited
therapeutically as will be appreciated by persons skilled in the
art. Such structures may also be found in the nucleic acid of
organisms having great importance in agriculture, pollution
control, industrial biochemistry, and otherwise. Accordingly,
pesticides, herbicides, fungicides, industrial organisms such as
yeast, bacteria, viruses, and the like, and biocatalytic systems
may be benefitted hereby.
[0064] In accordance with the present invention, there are provided
methods for the generation of virtual combinatorial libraries of
small molecules. These library molecules or members are generated
in silico. Library members of larger molecular weight, such as
those that are polymeric in nature, may also be generated using the
methods of the present invention.
[0065] The present invention further provides methods for tracking
and maintaining in databases, the fragments, reagents and unique
combinations of these used for the in silico generation of the
library members. Methods for interfacing the information necessary
for the generation of libraries in silico, as instructions designed
to direct the actual synthesis of the library members on an
instrument such as a parallel array synthesizer, are also provided
in the present invention.
[0066] The present invention also provides methods for the in
silico docking of the library members to identified target
molecules. According to these methods, individual library members
are allowed to bind to the desired target molecule in order to
identify those library members that demonstrate high affinity
binding to the targets.
[0067] In accordance with the present invention, there are provided
methods for the generation of virtual combinatorial libraries of
small molecules. These library molecules or members are generated
in silico. Library members of larger molecular weight, such as
those that are polymeric in nature, may also be generated using the
methods of the present invention.
[0068] The present invention further provides methods for tracking
and maintaining in databases, the fragments, reagents and unique
combinations of these used for the in silico generation of the
library members. Methods for interfacing the information necessary
for the generation of libraries in silico, as instructions designed
to direct the actual synthesis of the library members on an
instrument such as a parallel array synthesizer, are also provided
in the present invention.
[0069] The present invention is also directed to methods of
identifying compounds which bind to a molecular interaction site of
a nucleic acid comprising providing a numerical representation of
the three-dimensional structure of the molecular interaction site
and providing a compound data set comprising numerical
representations of the three dimensional structures of a plurality
of organic compounds. The numerical representation of the molecular
interaction site is then compared with members of the compound data
set to generate a hierarchy of organic compounds ranked in
accordance with the ability of the organic compounds to form
physical interactions with the molecular interaction site.
[0070] The present invention is also directed to data sets
comprising the numerical representations of the three dimensional
structures of molecular interaction sites and to the numerical
representations of the three dimensional structure of a plurality
of organic compounds.
[0071] The present invention is directed to methods of identifying
compounds which bind to a molecular interaction site of nucleic
acids. They comprise providing a numerical representation of the
three dimensional structure of the molecular interaction site,
providing a compound data set comprising numerical representations
of the three dimensional structures of a plurality of organic
compounds, comparing the numerical representation of the molecular
interaction site with members of the compound data set to generate
a hierarchy of organic compounds which is ranked in accordance with
the ability of the organic compounds to form physical interactions
with the molecular interaction site.
[0072] One aspect of the invention is a method to determine the
structure of biomolecular targets such as nucleic acids using mass
spectrometry. The method provides not only the primary, sequence
structure of nucleic acid targets, but also information about the
secondary and tertiary structure of nucleic acids, RNA and DNA,
including mismatched base pairs, loops, bulges, kinks, and stem
structures. This can be accomplished in accordance with one
embodiment by incorporating deoxynucleotide residues or other
modified residues into an oligoribonucleotide at specific sites
followed by selective cleavage of these hybrid RNA/DNA nucleic
acids in a mass spectrometer. It has now been found that
electrospray ionization of the nucleic acid, cleavage of the
nucleic acid, and subsequent tandem MS.sup.n spectrometry affords a
pattern of fragments that is indicative of the nucleic acid
sequence and structure. Cleavage is dependent on the sites of
incorporation of the deoxynucleotide or other foreign residues and
the secondary structure of the nucleic acid. This method therefore
provides mass spectral data that identifies the sites and types of
secondary structure present in the sequence of nucleic acids.
[0073] When the present methods are performed on a mixture of the
biomolecular target and a ligand or molecule that binds to the
target, it is possible to ascertain both the extent of interaction
and the location of this interaction between ligand and
biomolecule. The binding of the ligand to the biomolecule protects
the binding site on the biomolecule from facile cleavage during
mass spectrometry. Therefore, comparison of ESI-MS.sup.n mass
spectra generated, using this method, for RNA/DNA in the presence
and the absence of a binding ligand or drug reveals the location of
binding. This altered cleavage pattern is clearly discerned in the
mass spectrum and correlated to the sequence and structure of the
nucleic acid. Comparison of the abundance of the nucleic
acid-ligand noncovalent complex ion to the abundance of a similar
complex ion generated from a standard compound (such as paromomycin
for the 16S RNA A site ) whose binding affinity is known, allows
for the determination of relative binding affinity of the test
ligand.
[0074] The methods of this invention can be used for the rapid
screening of large collections of compounds. It is also possible to
screen mixtures of large numbers of compounds that are generated
via combinatorial or other means. When a large mixture of compounds
is exposed to a biomolecular target, such as a nucleic acid, a
small fraction of ligands may exhibit some binding affinity to the
nucleic acid. The actual number of ligands that may be detected as
binders is based on the concentration of the nucleic acid target,
the relative concentrations of the components of the combinatorial
mixture, and the relative binding affinities of these components.
The method is capable of separating different noncovalent
complexes, using techniques such as selective ion trapping, or
accumulation and analyzing each complex for the structure and
identity of the bound ligand using collisionally activated
dissociation or MS.sup.n experiments. The methods of this
invention, therefore, can not only serve as methods to screen
combinatorial libraries for molecules that bind to biomolecular
targets, but can also provide, in a straightforward manner, the
structural identity of the bound ligands. In this manner, any mass
redundancy in the combinatorial library does not pose a problem, as
the methods can provide high resolution molecular masses and also
able to discern differences between the different structures of
ligands of identical molecular mass using tandem methods.
[0075] In accordance with preferred embodiments, a target
biomolecule such as an RNA having a molecular interaction site, is
presented with one or more ligands or suspected ligands for the
interaction site under conditions such that interaction or binding
of the ligand to the molecular interaction site can occur. The
resulting complex, which may be of one or even hundreds of
individual complexes of ligands with the RNA or other biomolecule,
is then subjected to mass spectrometric evaluation in accordance
with the invention. "Preparative" mass spectrometry can isolate
individual complexes which can then be fragmented under controlled
conditions within the mass spectrometric environment for subsequent
analysis. In this way, the nature and degree of binding of the
ligands to the molecular interaction site can be ascertained.
Identification of specific, strong binding ligands can be made and
those selected for use either as therapeutics, agricultural,
industrial or other chemicals, or the same used as lead compounds
for subsequent modification into improved forms for such uses.
[0076] A further application of the present invention is the use of
mass spectrometric methods for the simultaneous screening of
multiple biomolecular targets against combinatorial libraries or
mixtures of compounds. This rather complex screening procedure is
made possible by the combined power of the mass spectrometric
methods used and the way in which the screening is performed. When
screening multiple target nucleic acids, for example, mass
redundancy is a concern, especially if two or more targets are of
similar sequence composition or mass. This problem is alleviated by
the present invention, by using special mass modifying, molecular
weight tags on the different nucleic acid targets being studied.
These mass modifying tags are typically large molecular weight,
non-ionic polymers including but not limited to, polyethylene
glycols, polyacrylamides and dextrans, that are available in many
different sizes and weights, and which may be attached at one or
more of many different possible sites on nucleic acids. Thus
similar nucleic acid targets may be differentially tagged and now
be readily differentiated, in the mass spectrum, from one another
by their distinctly different mass to charge ratios (m/z signals).
Using the methods of this invention, screening efforts can be
significantly accelerated because multiple targets can. now be
screened simultaneously against mixtures of large numbers of
compounds.
[0077] Another related advantage of the methods of this invention
is the ability to determine the specificity of binding interactions
between a new ligand and a biomolecular target. By simultaneously
screening a target nucleic acid, for example, and one or more
control nucleic acids against a combinatorial library or a specific
ligand, it is possible to ascertain, using the methods of this
invention, whether the ligand binds specifically to only the target
nucleic acids, or whether the binding observed with the target is
reproduced with control nucleic acids and is therefore
non-specific.
[0078] The methods of the invention are applicable to the study of
a wide variety of biomolecular targets that include, but are not
limited to, peptides, proteins, receptors, antibodies,
oligonucleotides, RNA, DNA, RNA/DNA hybrids, nucleic acids,
oligosaccharides, carbohydrates, and glycopeptides. The molecules
that may be screened by using the methods of this invention
include, but are not limited to, organic or inorganic, small to
large molecular weight individual compounds, mixtures and
combinatorial libraries of ligands, inhibitors, agonists,
antagonists, substrates, and biopolymers, such as peptides, nucleic
acids or oligonucleotides. The mass spectrometric techniques which
can be used in the methods of the invention include, but are not
limited to, MS.sup.n, collisionally activated dissociation (CAD)
and collisionally induced dissociation (CID) and infrared
multiphoton dissociation (IRMPD). A variety of ionization
techniques may be used including, but not limited to, electrospray,
MALDI and FAB. The mass detectors used in the methods of this
invention include, but are not limited to, FTICR, ion trap,
quadrupole, magnetic sector, time of flight (TOF), Q-TOF, and
triple quadrupole. The methods of this invention may also use
"hyphenated" techniques such as, but not limited to, LC/MS and
CE/MS, all as described more fully hereinafter.
[0079] While there are a number of ways to characterize binding
between molecular interaction sites and ligands, such as for
example, organic compounds, preferred methodologies are described
in U.S. patent applications filed on even date herewith and
assigned to the assignee of this invention. These application bear
U.S. Ser. Nos. ______ and have been assigned attorney docket
numbers IBIS-0002, IBIS-0003, IBIS-0004, IBIS-0005, and IBIS-0006.
All of the foregoing applications are incorporated by reference
herein in their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0080] FIG. 1 illustrates a flowchart comprising one preferred set
of method steps for identifying molecular interaction sites in
eukaryotic and prokaryotic RNA.
[0081] FIG. 2 is a flowchart describing a preferred set of
procedures in the Find Neighbors And Assemble ESTBlast
protocol.
[0082] FIG. 3 is a flowchart describing preferred steps in the
BlastParse protocol.
[0083] FIG. 4 is a flowchart describing preferred steps in the
Q-Compare protocol.
[0084] FIGS. 5A, 5B, and 5C illustrate flowcharts describing
preferred steps in the CompareOverWins protocol.
[0085] FIG. 6 is representative scatter plot of an interspecies
sequence comparison between mouse and human for a ferritin RNA.
[0086] FIG. 7 shows an example of self complementation analysis of
a single sequence.
[0087] FIG. 8 shows an overlay of self-complementarity plots of
certain orthologs, and selection for the most repetitive pattern in
each, resulting in a minimal number of possible folded
configurations as depicted in the diagonal strings of blocks.
[0088] FIG. 9 shows an exemplary descriptor.
[0089] FIG. 10 shows a set of e-value scores for ferritin.
[0090] FIG. 11 is a representative scatter plot of an interspecies
sequence comparison between human and trout for a ferritin RNA.
[0091] FIG. 12 is representative scatter plot of an interspecies
sequence comparison between human and chicken for a ferritin
RNA.
[0092] FIG. 13 shows a representative lookup table used in
Q-compare or CompareOverWins.
[0093] FIG. 14 shows a compound, compound CI, dissected into its
constituent fragments;
[0094] FIG. 15 shows the various identifying characteristics of the
fragments comprising compound CI;
[0095] FIG. 16 shows the various identifying characteristics of the
reagents used to introduce the corresponding fragments comprising
compound CI;
[0096] FIG. 17 is a list of transformations that link the fragments
and reagents associated with the generation of compound CI;
[0097] FIG. 18 is a schematic for the introduction of a common
fragment using two different reagents;
[0098] FIG. 19a is a schematic for the use of a single reagent for
the introduction of two different fragments into a compound;
[0099] FIG. 19b is a schematic showing the use of a common reagent
for the introduction of a common fragment into the compound which
can further be converted into two different fragments within the
compound generated;
[0100] FIG. 20 shows the symbolic addition of fragments yielding a
symbolic compound, compound CI';
[0101] FIG. 21 is a symbolic reagent table;
[0102] FIG. 22 is a symbolic fragment table;
[0103] FIG. 23 is a symbolic transformation table;
[0104] FIG. 24 shows the generation of individual compounds,
compounds C1 and C4, and a mixture, mixture M1;
[0105] FIG. 25 shows the generation of further mixture, mixture
M2;
[0106] FIG. 26 shows the generation of an additional mixture,
mixture M3;
[0107] FIGS. 27a and 27b show the generation of an additional
mixture, mixture M4;
[0108] FIG. 28 shows tables for tracking compound C1 by the
fragments added and or transformations performed;
[0109] FIG. 29 shows tables for tracking mixture M1 by the
transformations performed;
[0110] FIG. 30 shows tables for tracking mixture M2 by the
transformations performed; and
[0111] FIG. 31 shows tables for tracking mixture M3 by the
transformations performed.
[0112] FIG. 32 is a pictorial elevation view of an apparatus used
to robotically synthesize compound;
[0113] FIG. 33 is a pictorial plan view of an apparatus used to
robotically synthesize compounds;
[0114] FIG. 34 is a first synthetic reaction scheme for preparing a
library of compounds; and
[0115] FIG. 35 is a second synthetic reaction scheme for preparing
the library of compounds of FIG. 34.
[0116] FIG. 36 shows exemplary compounds which were docked to TAR
with subsequent evaluation of the solvation/desolvation energy.
[0117] FIG. 37 shows the target RNA for 4.5S--P48.
[0118] FIG. 38A shows a representative demonstration of
cap-dependent translation of three DNA plasmids with a wheat germ
lysate system: a) a luciferase gene with a 9 base leader sequence
before the AUG start codon; b) translation of a construct with the
TAR RNA structure adjacent to the cap; c) translation of a
construct with the TAR RNA structure separated from the cap by a 9
base leader sequence. Solid bars: no added m.sup.7G. Hatched bars:
added m.sup.7G.
[0119] FIG. 38B shows an exemplary inhibition of translation of an
mRNA construct containing the TAR RAN structure by a 39 amino acid
tat peptide: a) translation of a luciferase mRNA with a 9 base
leader sequence with and without 10 .mu.M added tat peptide; b)
translation of luciferase mRNA containing the TAR RNA structure
adjacent to the cap; c) translation of the luciferase/TAR RNA
construct with a 9 base leader in the presence/absence of 10 .mu.M
tat peptide.
[0120] FIG. 39 shows an exemplary dose-dependent inhibition of
translation of a luciferase mRNA construct containing a TAR RNA
structure in the 5'-UTR by ACD 00001199 (DecpBlue-3). Solid line:
inhibition of translation of the control luc+9 plasmid. Dashed
line: inhibition of expression of the luc+9 mRNA containing the TAR
RNA structure of the 5'-UTR.
[0121] FIG. 40 shows the sequence and structure of the 27-mer RNA
target corresponding to the 16S rRNA A-site.
[0122] FIG. 41 shows the ESI-CID-MS of a 27-mer RNA/DNA hybrid in
the presence and absence of paromomycin.
[0123] FIG. 42 shows the ESI-MS of a 27-mer RNA/DNA hybrid target
in the presence of paromomycin alone (panel a), and in the presence
of both paromomycin and a combinatorial library (panel b).
[0124] FIG. 43 shows the ESI-CID-MS spectrum of a combinatorial
library member-27mer RNA/DNA hybrid noncovalent complex ion of m/z
1919.0.
[0125] FIG. 44 shows the ESI-MS of a combinatorial library screened
against a 27mer RNA/DNA hybrid.
[0126] FIG. 45 shows the ESI-MS-MS analysis of the signal of m/z
1917.8 u arising from the binding of a member of mass 665 from
another combinatorial library.
[0127] FIG. 46 shows the ESI-MS-MS analysis of the signal of m/z
1934.3 u arising from the binding of a member of mass 720 from a
library.
[0128] FIGS. 47 and 48 show graphical representations of the
abundances of w and a-Base ions resulting from (CID) of ions from a
DNA:DNA duplex.
[0129] FIGS. 49, 50 and 51 depict MASS analyses to determine the
binding of ligands to a molecular interaction site.
[0130] FIG. 52 depicts high precision ESI-FTICR mass measurement of
the interaction of the 16S A site of an RNA complexed with
paromomycin.
[0131] As will be appreciated, the present invention provides for
the identification of molecules having the ability to modulate RNA
and other biomolecules. Novel combinations of procedures provide
extraordinary power and versatility to the present methods. While
it is preferred in some embodiments to integrate a number of
processes developed by the assignee of the present application as
will be set forth more fully herein, it should be recognized that
other methodologies may be integrated herewith to good effect.
Thus, while it is greatly advantageous to determine molecular
binding sited on RNAs and other molecules in accordance with the
teachings of this invention, the interactions of ligands and
libraries of ligands with RNA and other molecules identified as
being of interest may greatly benefit from other aspects of this
invention. All such combinations are within the spirit of the
invention.
[0132] In accordance with preferred embodiments, particular
structural elements in eukaryotic and prokaryotic nucleic acid,
molecular interaction sites, are identified. Thus, the present
invention is directed to methods of identifying particular
structural elements in eukaryotic and prokaryotic nucleic acid,
especially RNA molecules, which will interact with other molecules
to effect modulation of the RNA. "Modulation" refers to augmenting
or diminishing RNA activity or expression. The present invention is
outlined in flowchart form in FIG. 1. The structural elements in
eukaryotes and prokaryotes are referred to as "molecular
interaction sites." These elements contain secondary structure,
that is, have three-dimensional form capable of undergoing
interaction with "small" molecules and otherwise, and are expected
to serve as sites for interacting with "small" molecules, oligomers
such as oligonucleotides, and other compounds in therapeutic and
other applications.
[0133] Referring to FIG. 1, preferred steps for identifying
molecular interaction sites in target nucleic acids are shown in
the flow diagram. The nucleotide sequence of the target nucleic
acid is compared with the nucleotide sequences of a plurality of
nucleic acids from different taxonomic species, 10. The target
nucleic acid may be present in eukaryotic cells or prokaryotic
cells, the target nucleic acid may be bacterial or viral as well as
belonging to a "higher" organism such as human. Any type of nucleic
acid can serve as a target nucleic acid. Preferred target nucleic
acids include, but are not limited to, messenger RNA (mRNA),
pre-messenger RNA (pre-mRNA), transfer RNA (tRNA), ribosomal RNA
(rRNA), or small nuclear RNA (snRNA). Initial selection of a
particular target nucleic acid can be based upon any functional
criteria. Nucleic acids known to be important during inflammation,
cardiovascular disease, pain, cancer, arthritis, trauma, obesity,
Huntingtons, neurological disorders, or other diseases or
disorders, for example, are exemplary target nucleic acids.
[0134] Nucleic acids known to be involved in pathogenic genomes
such as, for example, bacterial, viral and yeast genomes are
exemplary prokaryotic nucleic acid targets. Pathogenic bacteria,
viruses and yeast are well known to those skilled in the art.
Exemplary nucleic acid targets are shown in Table 1. Applicants'
invention, however, is not limited to the targets shown in Table 1
and it is to be understood that the present invention is believed
to be quite general.
1TABLE 1 Exemplary RNA Targets Protein RNA Target GenBank #
Therapeutic 46 kD protein 3'-UTR stemloop X56134 cancer in vimentin
mRNA unknown-cGMP 5'-UTR of m10058 cancer regulated Asialoglyco-
protein receptor mRNA unknown unknown m11025 unknown unknown
insulin 3'-UTR of unknown inflammation regulated protein E-selectin
mRNA 30 kD protein 3'-UTR of lipo- m15856 obesity protein lipase
mRNA unknown 5'-UTR of NR2A U09002 trauma, paid, AD subunit of NMDA
receptor histone binding 3'-UTR of histone x57129 cancer protein
(HBP) mRNA + paralogs unknown 3'-UTR of p53 x02469 cancer mRNA p53
5'-UTR of mdm2 u39736 cancer oncogene mRNA unknown 5'-UTR of m27492
inflammation interleukin 1 type receptor (IL-1R1) none 5'-UTR of
muscle x84195 musculoskeletal acylphosphatase disease mRNA
ribosomal proteins 5'-UTR of c-myc V00568 cancer in multiple
myeloma unknown 5'-UTR of Huntingtons Huntingtons disease gene
unknown 5'-UTR of p30556 cardiovascular angiotensin AT disease
unknown zip code sequence d87468 unknown in ARC mRNA L-4 5'-UTR of
L4 d23660 cancer ribosomal protein L-32 5'-UTR of L32 x03342 cancer
ribosomal protein unknown TCTP, transla- x16064 cancer tionally
controlled tumor protein unknown 3'-UTR of B-F1- d00022 cancer
ATPase PU family of 3'-UTR of fem-3 X64962 unknown proteins, FBF in
C. elegans binding factor unknown 3'-UTR of x68505 metabolic
myocyte enhancer factor 2 MEF2A unknown 5'-UTR of k03195 diabetes
glucose trans- porter mRNA GLUT1 48 kD reticulocyte 3'-UTR of 15-
M23892 inflammation protein lipoxygenase La protein 5'-UTR of
cancer ribosomal RNA proteins unknown translational regu- S82692
inflammation lation of IL-2 unknown 3'-UTR of u81554 CNS CaMKIIa
mRNA in neurons bicoid (bcd) BRE 3'-UTR M21069 under fragment mRNA
development encoding cad protein 48/50 kD protein 3'-UTR structure
Y00443 cancer protamines 1 translin (human) protamine 1 Y00443
cancer TB-RBP (mouse) mRNA (human testes specific) translin (human)
protamine 2 X07862 unknown TB-RBP (mouse) mRNA translin (human)
transition protein x14474 cancer TB-RBP (mouse) mRNA translin
(human) Tau mRNA m13577 cancer TB-RBP (mouse) translin (human)
myelin basic x07948 cancer TB-RBP (mouse) protein mRNA p75 3'-UTR
of ribo- x59618 cancer nucleotide reductase R2 39 kD poly C alpha
globin v00493 cancer protein unknown beta protein v00497 metabolic
human Line-1 mRNA cancer, metabolic teratocarcinoma protein p40
RPL32 5'-UTR hairpin cancer structure in RPL32 Y-box proteins
family of tran- cancer scription factor mRNAs with a Y-box sequence
telomerase protein telomerase RNA AF015950 cancer ferritin,
transferrin IREs, internal inflammation loops in mRNA encoding
ferritin and transferrin ribosomal proteins 5'-UTR of M12873
inflammation PDGF2/c-sis mRNA zip code for 3'-UTR of beta cancer
localization actin unknown insulin 5'-UTR of x55362 cancer
regulated protein ornithine decarboxylase mRNA ribosomal proteins
ornithine cancer decarboxylase antizyme unknown FGF-5 inflammation
DFR protein factor 3'-UTR TGE X07384 cancer elements in the human
oncogene GLI DFR protein factor 3'-UTR tra-2 of unknown C. elegans
viral capsid protein 3'-UTR of alfalfa unknown mosaic virus RNA3
unknown BRE Bruno cancer response element in 3'-UTR of drosophila
oskar mRNA unknown NRE nanose cancer response element unknown
repeated element inflammation U1A RDB protein U1 snRNA inflammation
CD40 X60592 inflammation IGF-R X04434 inflammation M24599 A1
adenosine X68485 cardiovascular receptor B7-1 M27533 inflammation
B7-2 inflammation cyclophilin B M60857 inflammation M60457 M63573
cyclophilin C S71018 transplantation FKBP51 transplantation Th1
cytokines inflammation IFN .gamma. Th1 cytokines U03187
inflammation IL-12 NF-kappa B cancer ICAM-1 X06990 inflammation
L-selectin X16150 inflammation VCAM-1 M30257 inflammation Alpha 4
integrin X16983 inflammation X15356 Beta 7 U34971 inflammation
MadCAM-1 U43628 inflammation PECAM-1 M28526 inflammation LFA-1
Y00796 inflammation TACE inflammation LFA-3 X06296 inflammation
Y00636 CD-18 inflammation ICAM-3 X69819 inflammation ICAM-2 X15606
inflammation CD11a M87662 inflammation protein kinase C-.alpha.
cancer protein kinase C-.beta. X52479 cancer protein kinase
C-.delta. cancer protein kinase C-.epsilon. Z22521 cancer protein
kinase C-h X65293 cancer protein kinase C-m M55284 cancer protein
kinase C-.zeta. cancer unknown Z15108 unknown
[0135] Additional nucleic acid targets may be determied
inependently or can be selected from publicly available prokaryotic
and eukaryotic genetic databases known to those skilled in the art.
Preferred databases include, for example, Online Mendelian
Inheritance in Man (OMIM), the Cancer Genome Anatomy Project
(CGAP), GenBank, EMBL, PIR, SWISS-PROT, and the like. OMIM, which
is a database of genetic mutations associated with disease, was
developed, in part, for the National Center for Biotechnology
Information (NCBI). OMIM can be accessed through the Internet at,
for example, http://www.ncbi.nlm.nih.gov/Omim/. CGAP, which is an
interdisciplinary program to establish the information and
technological tools required to decipher the molecular anatomy of a
cancer cell. CGAP can be accessed through the Internet at, for
example, http://www.ncbi.nlm.nih.gov/ncicgap/. Some of these
databases may contain complete or partial nucleotide sequences. In
addition, nucleic acid targets can also be selected from private
genetic databases. Alternatively, nucleic acid targets can be
selected from available publications or can be determined
especially for use in connection with the present invention.
[0136] After a nucleic acid target is selected or provided, the
nucleotide sequence of the nucleic acid target is determined and
then compared to the nucleotide sequences of a plurality of nucleic
acids from different taxonomic species. In one embodiment of the
invention, the nucleotide sequence of the nucleic acid target is
determined by scanning at least one genetic database or is
identified in available publications. Preferred databases known and
available to those skilled in the art include, for example, the
Expressed Gene Anatomy Database (EGAD) and Unigene-Homo Sapiens
database (Unigene), GenBank, and the like. EGAD contains a
non-redundant set of human transcript (HT) sequences and can be
accessed through the Internet at, for example,
http://www.tigr.org/tdb- /egad/egad.html. Unigene is a system for
automatically partitioning GenBank sequences into a non-redundant
set of gene-oriented clusters. Each Unigene cluster contains
sequences that represent a unique gene, as well as related
information such as the tissue types in which the gene has been
expressed and map location.
[0137] In addition, Unigene contains hundreds of thousands of novel
expressed sequence tag (EST) sequences. Unigene can be accessed
through the Internet at, for example,
http://www.ncbi.nlm.nih.gov/UniGene/. These databases can be used
in connection with searching programs such as, for example, Entrez,
which is known and available to those skilled in the art, and the
like. Entrez can be accessed through the Internet at, for example,
http://www.ncbi.nlm.nih.gov/Entrez/. Preferably, the most complete
nucleic acid sequence representation available from various
databases is used. The GenBank database, which is known and
available to those skilled in the art, can also be used to obtain
the most complete nucleotide sequence. GenBank is the NIH genetic
sequence database and is an annotated collection of all publicly
available DNA sequences. GenBank is described in, for example, Nuc.
Acids Res., 1998, 26, 1-7, which is incorporated herein by
reference in its entirety, and can be accessed by those skilled in
the art through the Internet at, for example,
http://www.ncbi.nlm.nih.gov/Web/Genbank/index.html. Alternatively,
partial nucleotide sequences of nucleic acid targets can be used
when a complete nucleotide sequence is not available.
[0138] In another embodiment of the present invention, the
nucleotide sequence of the nucleic acid target is determined by
assembling a plurality of overlapping expressed sequence tags
(ESTs). The EST database (dbEST), which is known and available to
those skilled in the art, comprises approximately one million
different human mRNA sequences comprising from about 500 to 1000
nucleotides, and various numbers of ESTs from a number of different
organisms. dbEST can be accessed through the Internet at, for
example, http://www.ncbi.nlm.nih.gov/dbEST/index.htm- l. These
sequences are derived from a cloning strategy that uses cDNA
expression clones for genome sequencing. ESTs have applications in
the discovery of new genes, mapping of genomes, and identification
of coding regions in genomic sequences. Another important feature
of EST sequence information that is becoming rapidly available is
tissue-specific gene expression data. This can be extremely useful
in targeting selective gene(s) for therapeutic intervention. Since
EST sequences are relatively short, they must be assembled in order
to provide a complete sequence. Because every available clone is
sequenced, it results in a number of overlapping regions being
reported in the database.
[0139] Assembly of overlapping ESTs extended along both the 5' and
3' directions results in a full-length "virtual transcript." The
resultant virtual transcript may represent an already characterized
nucleic acid or may be a novel nucleic acid with no known
biological function. The Institute for Genomic Research (TIGR)
Human Genome Index (HGI) database, which is known and available to
those skilled in the art, contains a list of human transcripts.
TIGR can be accessed through the Internet at, for example,
http://www.tigr.org/. The transcripts were generated in this manner
using TIGR-Assembler, an engine to build virtual transcripts and
which is known and available to those skilled in the art.
TIGR-Assembler is a tool for assembling large sets of overlapping
sequence data such as ESTs, BACs, or small genomes, and can be used
to assemble eukaryotic or prokaryotic sequences. TIGR-Assembler is
described in, for example, Sutton, et al., Genome Science &
Tech., 1995, 1, 9-19, which is incorporated herein by reference in
its entirety, and can be accessed through the Internet at, for
example, ftp://ftp.tigr.org/pub/software/TIG- R assembler. In
addition, GLAXO-MRC, which is known and available to those skilled
in the art, is another protocol for constructing virtual
transcripts. In addition, "Find Neighbors and Assemble EST Blast"
protocol, which runs on a UNIX platform, has been developed by
Applicants to construct virtual transcripts. Preferred steps in the
Find Neighbors and Assemble EST Blast protocol is described in the
flowchart set forth in FIG. 2. PHRAP is used for sequence assembly
within Find Neighbors and Assemble EST Blast. PHRAP can be accessed
through the Internet at, for example,
http://chimera.biotech.washington.edu/uwgc/tools/phrap.htm. One
skilled in the art can construct source code to carry out the
preferred steps set forth in FIG. 2.
[0140] The nucleotide sequence of the nucleic acid target is
compared to the nucleotide sequences of a plurality of nucleic
acids from different taxonomic species. A plurality of nucleic
acids from different taxonomic species, and the nucleotide
sequences thereof, can be found in genetic databases, from
available publications, or can be determined especially for use in
connection with the present invention. In one embodiment of the
invention, the nucleic acid target is compared to the nucleotide
sequences of a plurality of nucleic acids from different taxonomic
species by performing a sequence similarity search, an ortholog
search, or both, such searches being known to perwons of ordinary
skill in the art.
[0141] The result of a sequence similarity search is a plurality of
nucleic acids having at least a portion of their nucleotide
sequences which are homologous to at least an 8 to 20 nucleotide
region of the target nucleic acid, referred to as the window
region. Preferably, the plurality of nucleotide sequences comprise
at least one portion which is at least 60% homologous to any window
region of the target nucleic acid. More preferably, the homology is
at least 70%. More preferably, the homology is at least 80%. Most
preferably, the homology is at least 90%. For example, the window
size, the portion of the target nucleotide to which the plurality
of sequences are compared, can be from about 8 to about 20,
preferably 10-15, most preferably about 11-12, contiguous
nucleotides. The window size can be adjusted accordingly. A
plurality of nucleic acids from different taxonomic species is then
preferably compared to each likely window in the target nucleic
acid until all portions of the plurality of sequences is compared
to the windows of the target nucleic acid. Sequences of the
plurality of nucleic acids from different taxonomic species which
have portions which are at least 60%, preferably at least 70%, more
preferably at least 80%, or most preferably at least 90% homologous
to any window sequence of the target nucleic acid are considered as
likely homologous sequences.
[0142] Sequence similarity searches can be performed manually or by
using several available computer programs known to those skilled in
the art. Preferably, Blast and Smith-Waterman algorithms, which are
available and known to those skilled in the art, and the like can
be used. Blast is NCBI's sequence similarity search tool designed
to support analysis of nucleotide and protein sequence databases.
Blast can be accessed through the Internet at, for example,
http://www.ncbi.nlm.nih.gov/BLAST/. The GCG Package provides a
local version of Blast that can be used either with public domain
databases or with any locally available searchable database. GCG
Package v9.0 is a commercially available software package that
contains over 100 interrelated software programs that enables
analysis of sequences by editing, mapping, comparing and aligning
them. Other programs included in the GCG Package include, for
example, programs which facilitate RNA secondary structure
predictions, nucleic acid fragment assembly, and evolutionary
analysis. In addition, the most prominent genetic databases
(GenBank, EMBL, PIR, and SWISS-PROT) are distributed along with the
GCG Package and are fully accessible with the database searching
and manipulation programs. GCG can be accessed through the Internet
at, for example, http://www.gcg.com/. Fetch is a tool available in
GCG that can get annotated GenBank records based on accession
numbers and is similar to Entrez. Another sequence similarity
search can be performed with GeneWorld and GeneThesaurus from
Pangea. GeneWorld 2.5 is an automated, flexible, high-throughput
application for analysis of polynucleotide and protein sequences.
GeneWorld allows for automatic analysis and annotations of
sequences. Like GCG, GeneWorld incorporates several tools for
homology searching, gene finding, multiple sequence alignment,
secondary structure prediction, and motif identification.
GeneThesaurus 1.0tm is a sequence and annotation data subscription
service providing information from multiple sources, providing a
relational data model for public and local data.
[0143] Another alternative sequence similarity search can be
performed, for example, by BlastParse. BlastParse is a PERL script
running on a UNIX platform that automates the strategy described
above. BlastParse takes a list of target accession numbers of
interest and takes each one through the preferred processes
described in the flowchart set forth in FIG. 3. BlastParse parses
all the GenBank fields into "tab-delimited" text that can then be
saved in a "relational database" format for easier search and
analysis, which provides flexibility. The end result is a series of
completely parsed GenBank records that can be easily sorted,
filtered, and queried against, as well as an annotations-relational
database.
[0144] Preferably, the plurality of nucleic acids from different
taxonomic species which have homology to the target nucleic acid,
as described above in the sequence similarity search, are further
delineated so as to find orthologs of the target nucleic acid
therein. An ortholog is a term defined in gene classification to
refer to two genes in widely divergent organisms that have sequence
similarity, and perform similar functions within the context of the
organism. In contrast, paralogs are genes within a species that
occur due to gene duplication, but have evolved new functions, and
are also referred to as isotypes. Optionally, paralog searches can
also be performed. By performing an ortholog search, an exhaustive
list of homologous sequences from as diverse organisms as possible
is obtained. Subsequently, these sequences are analyzed to select
the best representative sequence that fits the criteria for being
an ortholog. An ortholog search can be performed by programs
available to those skilled in the art including, for example,
Compare. Preferably, an ortholog search is performed with access to
complete and parsed GenBank annotations for each of the sequences.
Currently, the records obtained from GenBank are "flat-files", and
are not ideally suited for automated analysis. Preferably, the
ortholog search is performed using a Q-Compare program. Preferred
steps of the Q-Compare protocol are described in the flowchart set
forth in FIG. 4. The Blast Results-Relation database, depicted in
FIG. 3, and the Annotations-Relational database, depicted in FIG.
3, are used in the Q-Compare protocol, which results in a list of
ortholog sequences to compare in the interspecies sequence
comparisons programs described below.
[0145] The above-described similarity searches provide results
based on cut-off values, referred to as e-scores. E-scores
represent the probability of a random sequence match within a given
window of nucleotides. The lower the e-score, the better the match.
One skilled in the art is familiar with e-scores. The user defines
the e-value cut-off depending upon the stringency, or degree of
homology desired, as described above. In embodiments of the
invention where prokaryotic molecular interaction sites are
identified, it is preferred that any homologous nucleotide
sequences that are identified be non-human.
[0146] In another embodiment of the present invention, the
nucleotide sequences of a plurality of nucleic acids from different
taxonomic species are compared to the nucleotide sequence of the
target nucleic acid by performing a sequence similarity search
using dbEST, or the like, and constructing virtual transcripts.
Using EST information is useful for two distinct reasons. First,
the ability to identify orthologs for human genes in evolutionarily
distinct organisms in GenBank database is limited. As more effort
is directed towards identifying ESTs from these evolutionarily
distinct organisms, dbEST is likely to be a better source of
ortholog information.
[0147] Second, the attempt to sequence human genome is less than
10% complete. Thus, it is likely that the human dbEST will provide
more information for identifying primary targets as the sequence of
the human genome nears completion. EST sequences are short and need
to be assembled to be used. Preferably, a sequence similarity
search is performed using Smith-Waterman algorithms, as described
above, under high stringency against dbEST excluding human
sequences. Because dbEST contains sequencing errors, including
insertions and deletions, in order to accurately search for new
sequences, the search method used should allow for these gaps.
Because every available clone is sequenced, it results in a number
of overlapping regions being reported in the database. A
full-length or partial "virtual transcript" for non-human RNAs is
constructed by a process whereby overlapping EST sequences are
extended along both the 5' and 3' directions, until a "full-length"
transcript is obtained. In another embodiment of the invention, a
chimeric virtual transcript is constructed.
[0148] The resultant virtual transcript may represent an already
characterized RNA molecule or could be a novel RNA molecule with no
known biological function. As described above, TIGR HGI database
makes available an engine to build virtual transcripts called
TIGR-Assembler. GLAXO-MRC and GeneWorld from Pangea provide for
construction of virtual transcripts as well. As described above,
Find Neighbors and Assemble EST Blast can also be used to build
virtual transcripts.
[0149] Referring to FIG. 1, after the orthologs or virtual
transcripts described above are obtained through either the
sequence similarity search or the ortholog search, at least one
sequence region which is conserved among the plurality of nucleic
acids from different taxonomic species and the target nucleic acid
is identified, 20. Interspecies sequence comparisons can be
performed using numerous computer programs which are available and
known to those skilled in the art. Preferably, interspecies
sequence comparison is performed using Compare, which is available
and known to those skilled in the art. Compare is a GCG tool that
allows pair-wise comparisons of sequences using a window/stringency
criterion. Compare produces an output file containing points where
matches of specified quality are found. These can be plotted with
another GCG tool, DotPlot.
[0150] Alternatively, the identification of a conserved sequence
region is performed by interspecies sequence comparisons using the
ortholog sequences generated from Q-Compare in combination with
CompareOverWins, as described above. Preferably, the list of
sequences to compare, i.e., the ortholog sequences, generated from
Q-Compare, as described in FIG. 4, is entered into the
CompareOverWins algorithm. Preferred steps in the CompareOverWins
are described in FIGS. 5A, 5B, and 5C. Preferably, interspecies
sequence comparisons are performed by a pair-wise sequence
comparison in which a query sequence is slid over a window on the
master target sequence. Preferably, the window is from about 10 to
about 30 contiguous nucleotides. More preferably, the window is 21
nucleotides. If the number of identical bases (matches) within this
window reaches a user-defined threshold, a score is given.
[0151] Sequence homology between the window sequence of the target
nucleic acid and the query sequence of any of the plurality of
nucleic acid sequences obtained as described above, is preferably
at least 60%, more preferably at least 70%, more preferably at
least 80%, and most preferably at least 90%. This process is
repeated until every base on the query nucleic acid, which is a
member of the plurality of nucleic acids described above, has been
compared to every base on the master target sequence. The resulting
scoring matrix can be plotted as a scatter plot. Based on the match
density at a given location, there may be no dots, isolated dots,
or a set of dots so close together that they appear as a line. The
presence of lines, however small, indicates primary sequence
homology. A representative scatter plot of such interspecies
sequence comparison is depicted in FIG. 6. Sequence conservation
within nucleic acid molecules, particularly the UTRs of RNA, in
divergent species is likely to be an indicator of conserved
regulatory elements that are also likely to have a secondary
structure. The results of the interspecies sequence comparison can
be analyzed using MS Excel and visual basic tools in an entirely
automated manner as known to those skilled in the art.
[0152] Referring to FIG. 1, after at least one region that is
conserved between the nucleotide sequence of the nucleic acid
target and the plurality of nucleic acids from different taxonomic
species, preferably via the orthologs, is identified, the conserved
region is analyzed to determine whether it contains secondary
structure, 30. Determining whether the identified conserved regions
contain secondary structure can be performed by a number of
procedures known to those skilled in the art. Determination of
secondary structure is preferably performed by self complementarity
comparison, alignment and covariance analysis, secondary structure
prediction, or a combination thereof.
[0153] In one embodiment of the invention, secondary structure
analysis is performed by alignment and covariance analysis.
Numerous protocols for alignment and covariance analysis are known
to those skilled in the art. Preferably, alignment is performed by
ClustalW, which is available and known to those skilled in the art.
ClustalW is a tool for multiple sequence alignment that, although
not a part of GCG, can be added as an extension of the existing GCG
tool set and used with local sequences. ClustalW can be accessed
through the Internet at, for example,
http://dot.imgen.bcm.tmc.edu:9331/multi-align/Options/clustalw.html.
ClustalW is also described in Thompson, et al., Nuc. Acids Res.,
1994, 22, 4673-4680, which is incorporated herein by reference in
its entirety. These processes can be scripted to automatically use
conserved UTR regions identified in earlier steps. Seqed, a UNIX
command line interface available and known to those skilled in the
art, allows extraction of selected local regions from a larger
sequence. Multiple sequences from many different species can be
clustered and aligned for further analysis.
[0154] Covariation is a process of using phylogenetic analysis of
primary sequence information for consensus secondary structure
prediction. Covariation is described in the following references,
each of which is incorporated herein by reference in their
entirety: Gutell, et al., "Comparative Sequence Analysis Of
Experiments Performed During Evolution" In Ribosomal RNA Group I
Introns, Green, Ed., Austin:Landes, 1996; Gautheret, et al., Nuc.
Acids Res., 1997, 25, 1559-1564; Gautheret, et al., RNA, 1995, 1,
807-814; Lodmell, et al., Proc. Natl. Acad. Sci. USA, 1995, 92,
10555-10559; Gautheret, et al., J. Mol. Biol., 1995, 248, 27-43;
Gutell, Nuc. Acids Res., 1994, 22, 3502-3517; Gutell, Nuc. Acids
Res., 1993, 21, 3055-3074; Gutell, Nuc. Acids Res., 1993, 21,
3051-3054; Woese, Proc. Natl. Acad. Sci. USA, 1989, 86, 3119-3122;
and Woese, et al., Nuc. Acids Res., 1980, 8, 2275-2293. Preferably,
covariance software is used for covariance analysis. Preferably,
Covariation, a set of programs for the comparative analysis of RNA
structure from sequence alignments, is used. Covariation uses
phylogenetic analysis of primary sequence information for consensus
secondary structure prediction. Covariation can be obtained through
the Internet at, for example,
http://www.mbio.ncsu.edulRNaseP/info/programs/programs.html. A
complete description of a version of the program has been published
(Brown, J. W. 1991 Phylogenetic analysis of RNA structure on the
Macintosh computer. CABIOS7:391-393). The current version is v4.1,
which can perform various types of covariation analysis from RNA
sequence alignments, including standard covariation analysis, the
identification of compensatory base-changes, and mutual information
analysis. The program is well-documented and comes with extensive
example files. Compiled as a stand-alone program; it does not
require Hypercard (although a much smaller `stack` version is
included). This program will run in any Macintosh environment
running MacOS v7.1 or higher. Faster processor machines (68040 or
PowerPC) is suggested for mutual information analysis or the
analysis of large sequence alignments.
[0155] In another embodiment of the invention, secondary structure
analysis is performed by secondary structure prediction. There are
a number of algorithms that predict RNA secondary structures based
on thermodynamic parameters and energy calculations. Preferably,
secondary structure prediction is performed using either M-fold or
RNA Structure 2.52. M-fold can be accessed through the Internet at,
for example, http://www.ibc.wustl.edu/-zuker/ma/form2.cgi or can be
downloaded for local use on UNIX platforms. M-fold is also
available as a part of GCG package. RNA Structure 2.52 is a windows
adaptation of the M-fold algorithm and can be accessed through the
Internet at, for example,
http://128.151.176.70/RNAstructure.html.
[0156] In another embodiment of the invention, secondary structure
analysis is performed by self complementarity comparison.
Preferably, self complementarity comparison is performed using
Compare, described above. More preferably, Compare can be modified
to expand the pairing matrix to account for G-U or U-G basepairs in
addition to the conventional Watson-Crick G-C/C-G or A-U/U-A pairs.
Such a modified Compare program (modified Compare) begins by
predicting all possible base-pairings within a given sequence. As
described above, a small but conserved region, preferably a UTR, is
identified based on primary sequence comparison of a series of
orthologs. In modified Compare, each of these sequences is compared
to its own reverse complement. FIG. 7 depicts an exemplary self
complementarity analysis. Allowable base-pairings include
Watson-Crick A-U, G-C pairing and non-canonical G-U pairing. An
overlay of such self complementarity plots of all available
orthologs, and selection for the most repetitive pattern in each,
results in a minimal number of possible folded configurations. FIG.
8 shows an exemplary overlay. These overlays can then used in
conjunction with additional constraints, including those imposed by
energy considerations described above, to deduce the most likely
secondary structure.
[0157] A result of the secondary structure analysis described
above, whether performed by alignment and covariance, self
complementarity analysis, secondary structure predictions, such as
using M-fold or otherwise, is the identification of secondary
structure in the conserved regions among the target nucleic acid
and the plurality of nucleic acids from different taxonomic
species, 40. Exemplary secondary structures that may be identified
include, but are not limited to, bulges, loops, stems, hairpins,
knots, triple interacts, cloverleafs, or helices, or a combination
thereof. Alternatively, new secondary structures may be
identified.
[0158] In another embodiment of the invention, once the secondary
structure of the conserved region has been identified, as described
above, at least one structural motif for the conserved region
having secondary structure is identified. These structural motifs
correspond to the identified secondary structures described above.
For example, analysis of secondary structure by self
complementation may provide one type of secondary structure,
whereas analysis by M-fold may provide another secondary structure.
All the possible secondary structures identified by secondary
structure analysis described above are, thus, represented by a
family of structural motifs.
[0159] Once the secondary structure(s) of the target nucleic acids,
as well as the secondary structures of nucleic acids from different
taxonomic species, have been identified, further nucleic acids can
be identified by searching on the basis of structure, rather than
by primary nucleotide sequence, as described above. Additional
nucleic acids which have secondary structure similar or identical
to the secondary structure found as described above can be
identified by constructing a family of descriptor elements for the
structural motifs described above, and identifying other nucleic
acids having secondary structures corresponding to the descriptor
elements. The combination of any or all of the nucleic acids having
secondary structure can be compiled into a database. The entire
process can be repeated with a different target nucleic acid to
generate a plurality of different secondary structure groups which
can be compiled into the database. Thus, databases of molecular
interaction sites can be compiled by performing by the invention
described herein.
[0160] After the hypothetical structure motifs are determined from
the secondary structure analysis described above, a family of
structure descriptor elements is constructed. Preferably, the
structural motifs described above are converted into a family of
descriptor elements. An exemplary descriptor element is shown in
FIG. 9. One skilled in the art is familiar with construction of
descriptors. Structure descriptors are described in, for example,
Laferriere, et al., Comput. Appl. Biosci., 1994, 10, 211-212,
incorporated herein by reference in its entirety. A different
structure descriptor element is constructed for each of the
structural motifs identified from the secondary structure analysis.
Briefly, the secondary structure is converted to a generic text
string, such as shown in FIG. 9. For novel motifs, further
biochemical analysis such as chemical mapping or mutagenesis may be
needed to confirm structure predictions. Descriptor elements may be
defined to have various stringency.
[0161] For example, referring to FIG. 9, the region termed Hi,
which comprises the first region of the stem, can be described as
NNN:NNN, which contemplates any complementary base pairing
including G-C, C-G, A-U, and U-A. The Hi region may also be
designated so as to include only C-G or A-U, etc., base pairing. In
addition, the descriptor elements can be defined to allow for a
wobble. Thus, descriptor elements can be defined to have any level
of stringency desired by the user. Applicants' invention, thus, is
also directed to a database comprising different descriptor
elements.
[0162] After a family of structure descriptor elements is
constructed, nucleic acids having secondary structure which
correspond to the structure descriptor elements are identified.
Preferably, nucleic acids having secondary structure which
correspond to the structure descriptor elements are identified by
searching at least one database, performing clustering and
analysis, identifying orthologs, or a combination thereof. Thus,
the identified nucleic acids have secondary structure which falls
within the scope of the secondary structure defined by the
descriptor elements. Thus, the identified nucleic acids have
secondary structure identical to nearly identical, depending on the
stringency of the descriptor elements, to the target nucleic
acid.
[0163] In one embodiment of the invention, nucleic acids having
secondary structure which correspond to the structure descriptor
elements are identified by searching at least one database. Any
genetic database can be searched. Preferably, the database is a UTR
database, which is a compilation of the untranslated regions in
messenger RNAs. A UTR database is accessible through the Internet
at, for example, ftp://area.ba.cnr.it/pub/embnet/database/utr/.
Preferably the database is searched using a computer program, such
as, for example, Rnamot, a UNIX-based motif searching tool
available from Daniel Gautheret. Each "new" sequence that has the
same motif is then queried against public domain databases to
identify additional sequences. Results are analyzed for recurrence
of pattern in UTRs of these additional ortholog sequences, as
described below, and a database of RNA secondary structures is
built. One skilled in the art is familiar with Rnamot. Briefly,
Rnamot takes a descriptor string, such as the one shown in FIG. 9,
and searches any Fasta format database for possible matches.
Descriptors can be very specific, to match exact nucleotide(s), or
can have built-in degeneracy. Lengths of the stem and loop can also
be specified. Single stranded loop regions can have a variable
length. G-U pairings are allowed and can be specified as a wobble
parameter. Allowable mismatches can also be included in the
descriptor definition. Functional significance is assigned to the
motifs if their biological role is known based on previous
analysis. Known regulatory regions such as Iron Response Element
have been found using this technique (see, Example 1 below). In
embodiments of the invention in which a database containing
prokaryotic molecular interaction sites is compiled, it is
preferable to refrain from searching human sequences or,
alternatively, discarding human sequences when found.
[0164] In another embodiment of the invention, the nucleic acids
identified by searching databases such as, for example, searching a
UTR database using Rnamot, are clustered and analyzed so as to
determine their location within the genome. The results provided by
Rnamot simply identify sequences containing the secondary structure
but do not give any indication as to the location of the sequence
in the genome. Clustering and analysis is preferably performed with
ClustalW, as described above.
[0165] In another embodiment of the invention, after clustering and
analysis is performed as described above, orthologs are identified
as described above. However, in contrast to the orthologs
identified above, which were solely identified on the basis of
their primary nucleotide sequences, these new orthologous sequences
are identified on the basis of structure using the nucleic acids
identified using Rnamot. Identification of orthologs is preferably
performed by BlastParse or Q-Compare, as described above. In
embodiments of the invention in which a database containing
prokaryotic molecular interaction sites is compiled, it is
preferable to refrain from finding human orthologs or,
alternatively, discarding human orthologs when found.
[0166] After nucleic acids having secondary structures which
correspond to the structure descriptor elements are identified, any
or all of the nucleotide sequences can be compiled into a database
by standard compiling protocols known to those skilled in the art.
One database may contain eukaryotic molecule interaction sites and
another database may contain prokaryotic molecule interaction
sites.
[0167] The present invention is also directed to oligonucleotides
comprising a molecular interaction site that is present in the RNA
of a selected organism and in the RNA of at least one preferably
several additional organisms. The nucleotide sequence of the
oligonucleotide is selected to provide the secondary structure of
the molecular interaction sites described above. The nucleotide
sequence of the oligonucleotide is preferably the nucleotide
sequence of the target nucleic acids described above.
Alternatively, the nucleotide sequence is preferably the nucleotide
sequence of nucleic acid from a plurality of different taxonomic
species which also contain the molecular interaction site. The
molecular interaction site serves as a binding site for at least
one molecule which, when bound to the molecular interaction site,
modulates the expression of the RNA in the selected organism.
[0168] The present invention is also directed to oligonucleotides
comprising a molecular interaction site that is present in a
prokaryotic RNA and in at least one additional prokaryotic RNA,
wherein the molecular interaction site serves as a binding site for
at least one molecule which, when bound to the molecular
interaction site, modulates the expression of the prokaryotic RNA.
The additional organism is selected from all all eukaryotic and
prokaryotic organisms and cells but is not the same organism as the
selected organism. Oligonucleotides, and modifications thereof, are
well known to those skilled in the art. The oligonucleotides of the
invention can be used, for example, as research reagents to detect,
for example, naturally occurring molecules which bind the molecular
interaction sites. The oligonucleotides of the invention can also
be used as decoys to compete with naturally-occurring molecular
interaction sites within a cell for research, diagnostic and
therapeutic applications. Molecules which bind to the molecular
interaction site modulate, either by augmenting or diminishing, the
expression of the RNA. The oligonucleotides can also be used in
agricultural, industrial and other applications.
[0169] The present invention is also directed to pharmaceutical
compositions comprising the oligonucleotides described above in
combination with a pharmaceutical carrier. A "pharmaceutical
carrier" is a pharmaceutically acceptable solvent, diluent,
suspending agent or any other pharmacologically inert vehicle for
delivering one or more nucleic acids to an animal, and are well
known to those skilled in the art. The carrier may be liquid or
solid and is selected, with the planned manner of administration in
mind, so as to provide for the desired bulk, consistency, etc.,
when combined with the other components of a pharmaceutical
composition. Typical pharmaceutical carriers include, but are not
limited to, binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.);
fillers (e.g., lactose and other sugars, microcrystalline
cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose,
polyacrylates or calcium hydrogen phosphate, etc.); lubricants
(e.g., magnesium stearate, talc, silica, colloidal silicon dioxide,
stearic acid, metallic stearates, hydrogenated vegetable oils, corn
starch, polyethylene glycols, sodium benzoate, sodium acetate,
etc.); disintegrates (e.g., starch, sodium starch glycolate, etc.);
or wetting agents (e.g., sodium lauryl sulphate, etc.).
[0170] The present invention is directed to computational methods
employed for the in silico design and synthesis of combinatorial
libraries of small molecules. The library members are generated in
silico. The present invention also encompasses methods for tracking
and storing the information generated during the in silico creation
of library members into relational databases for later access and
use. For the purposes of this specification, in silico refers to
the creation in a computer memory, i.e., on a silicon or other like
chip. Stated otherwise in silico means "virtual."
[0171] According to the methods of the present invention, each
compound or library member is dissected into its component or
constituent parts referred to as fragments. Thus each compound that
is generated is considered to be comprised of constituent fragments
such that the sum of the molecular formulas of each of the
fragments when added together totals the molecular formula of the
compound generated. This dissection can be done in a variety of
ways using chemical intuition. Thus a variety of components of
fragments may be identified, each of which lend themselves to
readily available reagents or reactions to generate diverse
compounds. Further, each fragment is associated with at least one
reagent, which represents the necessary chemical to be used to
introduce that desired fragment into the compound being generated
in silico. Dissection of compounds is based on the ease of
synthesis of the reagents, commercial availability of the reagents,
or a combination of both. Each of the fragments and reagents are
stored in a relational database and are described in terms of
identifying characteristics in the database. A fragment may be
available from a variety of starting materials or reaction schemes.
So when a library is being generated, which entails building a
database, the fragments used in building that library can be stored
in the database using the corresponding set of reagents and
reaction conditions. When another library is to be generated, the
fragment information stored in the database is now available for
use in the generation of the new library of compounds. Similarly,
when a third library is being generated, an even greater quantity
of fragment, reagent, and reaction information is available in the
database. Thus the methods of the present invention represent a
dynamic method of building a database associated with building
libraries of compounds. Initial library generation requires
database input for fragments, reagents and transformations
necessary for desired library. As the database grows, however, an
increasing number of fragments and reagents are available in the
database, which simplifies the generation of subsequent libraries
of compounds and makes for more routine combinatorial synthetic
efforts which can be accomplished with increasing ease and
efficacy.
[0172] Fragments that are recorded in the database may be defined
using identifying characteristics. Identifying characteristics
defining fragments include a structural representation (as a
2-dimensional or 3-dimensional file), name, molecular weight,
molecular formula, and attachment points or nodes (which denote
sites of attachment or linkage of the fragment to other fragments
of the compound being generated in silico). For the purpose of
describing this invention, 2-dimensional representations are used,
which are further simplified by the use of symbolic representations
without reference to any particular chemical entities. The symbolic
representations as used herein merely shows how fragments can be
tracked to further the methods of the present invention. Other
identifying characteristics may also be added to the database. Any
characteristic that is desired to be tracked may be included in the
database, including biological data, chemical reactivity rates, or
other physical or chemical properties. Further, a fragment may also
be created by modifying a reagent, and such modifications can be
added to the database in terms of changes made to the reagent
structure. Some of the identifying characteristics associated with
any fragment may be common to those of the corresponding reagent.
The related fragment thus created can then be stored in the
relational database.
[0173] Identifying characteristics defining reagents include a
structural representation, name, molecular weight, molecular
formula, and source, such as a commercial source or a unique
compound defined by the user. In case of a commercial source for
the reagent, a catalog number or a link to a web page can be
provided. Some commonalities may exist between the identifying
characteristics associated with a reagent and those associated with
the related fragment.
[0174] Further, in accordance with the present invention, a
compound is the sum of various transformations. Transformation is
the nomenclature attributed according to the present invention to a
chemical synthesis. A transformation is a 1:1 link between a
fragment and a reagent. Thus each transformation describes a unique
conversion of a reagent into the corresponding fragment as
introduced into a compound. When the compound being generated in
silico is broken down into its component fragments, and the
corresponding reagents have been identified, each fragment is
linked to the corresponding reagent in a 1:1 relationship in order
to describe a transformation. Thus, according to the present
invention, a transformation may be viewed as the source of a
fragment, thereby linking that fragment to a particular synthetic
method or reaction. This description of a transformation according
to the methods of the present invention also includes any auxiliary
reagents or conditions used to effect the reaction denoted by the
transformation, such as temperature and pressure requirements,
catalysts, activators, solvents, or other additives.
[0175] Each combination of a fragment and reagent in a 1:1 link
comprises a different transformation. Therefore, each
transformation is unique. The present invention allows the tracking
of fragments in terms of the reaction or transformation in which
those fragments are introduced into the compounds of the library.
Thus the database describes not only the compounds generated in
terms of their constituent fragments, but also in terms of the
synthetic pathways to produce those compounds, i.e. the related
transformations to generate the library compounds. In this manner,
a user of the present invention can generate a virtual library of
compounds by simply selecting the fragments desired. Alternately, a
user can also generate the compounds by selecting the chemical
pathways required for actual synthesis of the compounds. This is
accomplished by selecting the appropriate transformation associated
with the generation of the desired compounds. Here, the user uses
intuition or an in silico expert system to assist in selecting
those transformations that are expected to allow generation or
synthesis of the desired compounds. Each of the transformations
created in silico is stored in the relational database and
described in terms of identifying characteristics. Identifying
characteristics defining transformations include the fragment, the
reagent, and any auxiliary reagent or conditions necessary to
effect the conversion of the reagent into the fragment as
incorporated into the compound.
[0176] For example, consider in FIG. 14 the in silico generation of
compound CI according to the methods of the present invention. As
shown in FIG. 14, upon dissection of CI (molecular formula of
C.sub.12H.sub.18N.sub.2O.sub.5S.sub.1), its constituent fragments
can be denoted as F.sub.i (molecular formula of H.sub.2NO),
F.sub.ii (molecular formula of C.sub.5H.sub.9NO), and F.sub.iii
(molecular formula of C.sub.7H.sub.7O.sub.3S). F.sub.i can also be
a hydroxyl amine moiety linked to a solid support, i.e. P--O--NH,
wherein P is a solid support. The sum of the molecular formulas of
each of the fragments totals the molecular formula of compound
CI.
[0177] As shown in FIG. 15, each of the fragments, F.sub.i,
F.sub.ii, and F.sub.iii, are stored in a relational database, and
are described in terms of identifying characteristics including a
structural representation (which may be 2-dimensional or
3-dimensional), an identifier or name, molecular formula and
attachment points or nodes which signify sites on the fragment
which are linked to other fragments in compound CI. Other
information such as molecular weight can also be associated with
the fragment in the database.
[0178] As shown in FIG. 16, each of the corresponding reagents
(R.sub.i, R.sub.ii, and R.sub.iii) are also stored in the
relational database, and described in terms of identifying
characteristics. Identifying characteristics used to define the
reagents include a structural representation, and identifier or
name and molecular formula. As with the fragment, other associated
information such as molecular weight and source (such as a
commercial source verses user-supplied, amount on hand, special
handling, etc.) can also be stored in database in association with
the individual reagents.
[0179] Next, each of the transformations associated with the in
silico generation of compound CI are also stored in the relational
database. As shown in FIG. 17, transformation T.sub.i links reagent
R.sub.i with fragment F.sub.i, T.sub.ii links R.sub.ii with
F.sub.ii, and T.sub.iii links R.sub.iii with F.sub.iii in a 1:1
relationship. Also, associated with each transformation is the
necessary reaction condition, so that transformation T.sub.i is
associated with reaction condition alpha, T.sub.ii with reaction
condition beta, and T.sub.iii with reaction condition gamma. In the
case of transformation T.sub.iii, reagent R.sub.iii may be a
hydroxyl amine attached to a solid support so that fragment
F.sub.iii can be represented as a hydroxyl amine moiety attached to
a solid support.
[0180] While each fragment may be arrived at or generated by a
unique corresponding reagent, the present invention also
encompasses common fragments that may be generated via two or more
reagents, so that two or more transformations can lead to the same
fragment. As shown in FIG. 18, the common fragment
CH.sub.3--CH.sub.2--C(.dbd.O)-- may be arrived at via
transformation A, which employs reagent X (an acid chloride),
CH.sub.3--CH.sub.2--C(.dbd.O)Cl. The common fragment can also be
introduced into a compound being generated in silico via
transformation B, which employs reagent Y (an acid anhydride),
CH.sub.3--CH.sub.2--C(.db- d.O)--O--C(.dbd.O)--CH.sub.2--CH.sub.3.
Therefore, in accordance with the methods of the present invention,
a common fragment can be introduced into the compound via two or
more different reagents, and thus via two or more distinct
transformations.
[0181] Alternately, a common reagent may be employed to effect two
or more conversions forming two or more different fragments. This
then represents two or more different transformations associated
with different conditions. For example, as shown in FIG. 19a,
common reagent Z, CH.sub.3--CH.sub.2--NH.sub.2, can be employed to
introduce an alkene fragment into the compound under conditions
favoring Schiff's base formation. This represents transformation X.
The same common reagent Z, however, can also be employed to
introduce an amide fragment into the compound by using a different
set of conditions, constituting transformation Y. Thus, a common
reagent can introduce two or more different fragments into final
compounds being generated in silico, and can be associated with two
or more transformations depending upon the conditions associated
with each of those transformations.
[0182] Additionally, once a fragment has been introduced into a
compound, it can be further modified and converted into yet another
fragment without effecting any other chemical changes within the
compound formed. As an example, shown in FIG. 19b, consider common
reagent Z', CH.sub.3--CH.sub.2--C(.dbd.O)CH.sub.2--Cl. Common
reagent Z' corresponds to a fragment having the structure
CH.sub.3--CH.sub.2--C(.dbd.O)CH.sub.2-- -. Common reagent Z' may be
used to introduce an alkene fragment into the final compound,
representing transformation X', under conditions favoring reduction
and dehydration. Common reagent Z', however, can also be used to
introduce a hydroxyalkyl fragment into the final compound under
conditions favoring reduction. This represents transformation
Y'.
[0183] The present invention may be described more generally, in
terms of symbolic representations. Symbolic representations are
used to describe the methods of the present invention because such
representations are not limited to any particular chemistry.
Symbolic representations merely denote the manner of using the
present invention with multiple chemical entities. Each symbol used
in the representations describing the present invention may
represent one compound or multiple compounds because the present
invention is not limited to tracking a single compound, but may be
used to track a vast variety of compounds that can be
generated.
[0184] FIG. 20 shows the symbolic addition of fragments which
yields compound CI'. The fragments have structures F.sub.i',
F.sub.ii', and F.sub.iii' that are added sequentially to yield
compound CI'. Structures F.sub.i', F.sub.ii', and F.sub.iii' are
symbolic representations of the fragments that constitute compound
CI'. These fragments can be stored in the relational database with
the corresponding identifying characteristics for each of them,
including the structural representation, name, molecular formula,
and attachment sites or nodes. A visual inspection of compounds C1
and C1' revels the commonality between the chemical compound C1 and
the symbolic representation of a compound C1' as well as the
chemical structure of the fragments and the symbolic structure of
the fragments.
[0185] A symbolic reagent table is shown in FIG. 21. Reagents R1 to
R10 can be described in terms of their structure, name, molecular
formula, molecular weight, and source as well as other information
that might be desired to be associated with the reagents. R3 and R4
are two different reagents, but may be used to introduce the same
fragment into a compound. This depends upon the reaction conditions
used as reagent R3 is used in a transformation associated with one
set of conditions, while reagent R4 is used in another
transformation associated with a different set of conditions. Also,
reagent R5 is comprised of a mixture of two reagents or components.
These may be (R)- and (S)-stereoisomers, D- and L-isomers, or may
be two completely different reagents. While R5 here is represented
as a mixture of only two reagents or components, it will be
recognized by the art-skilled that the methods of the present
invention may be practiced using a mixture of two or more reagents.
Typical reagent mixtures used in constructing libraries might have
four, five or more individual reagent constituting the mixture.
[0186] FIG. 22 shows a symbolic fragment table. Fragments F1 to F8
are stored in the relational database with identifying
characteristics that include a structural representation, name,
molecular weight, molecular formula, and attachment sites or nodes.
This table depicts symbolic representations of the various
fragments that are introduced into the compounds of the library by
the use of reagents symbolized in FIG. 21. Thus it can be seen that
fragment F1 can be introduced into the compound by employing
reagent R1. In fragment F1, X is an identifier for an attachment
site. This indicates that X is the site at which F1 attaches to
another fragment in a compound. Similarly, fragment F2 may be
introduced into a compound (attaching at its X site) by employing
reagent R2.
[0187] Fragment F3, however, can be introduced into the compound by
the use of either reagent R3 or R4. This allows for selection in
the choice of the reagent used, and also allows for the
consideration of the compatibility of the chemistries involved in
the introduction of other fragments into the compound. Next,
fragment F4 (which is a mixture of fragments) can be introduced via
the use of reagent R5, which is a mixture of reagents, as shown in
FIG. 21.
[0188] Fragment F5 has two attachment sites, indicating that other
fragments can attach at sites X and Y when F5 has been incorporated
into a compound. The presence of two attachment sites indicates
that two attachments may be undertaken to build a compound when
dealing with F5. Here again, as before, F5 can be introduced into
the compound using either of reagents R6 or R7, depending upon the
reaction conditions used and the chemistries involved when
introducing other fragments to build the compound.
[0189] Fragments F7 and F8 can be introduced into a compound being
created in silico by employing reagents R9 and R10, respectively.
Both these fragments have three attachment sites, indicating that
three attachments to other fragments can occur when using these
fragments to build a compound in silico. While fragments F7 and F8
have three attachment sites, it is recognized by the art-skilled
that more than three attachment sites may be present in a fragment,
allowing for more attachments to the fragment upon introduction
into a compound (with the use of an appropriate reagent).
[0190] With the fragment and reagent tables in place in the
relational database, a transformation table is created in
accordance with the methods of the present invention, by linking a
fragment with a reagent to form a unique transformation. FIG. 23
shows a symbolic transformation table where a fragment is linked to
a reagent in a 1:1 relationship.
[0191] The identifying characteristics describing each
transformation include a 1:1 link (a one to one link) between a
fragment and a reagent, and the reaction conditions which include,
solvent, concentration, temperature and pressure requirements, or
auxiliary reagents necessary to effect the introduction of the
fragment into the compound by using an appropriate reagent.
Auxiliary reagents include catalysts, activators, acids, bases or
other chemicals or additives necessary to effect the fragment
introduction described. For example a base can always be added with
an alkyl halide to scavenge the acid generated with use of the
alkyl halide.
[0192] As seen in FIG. 23, transformation T1 links fragment F1 with
reagent R1. T1 also specifies the reaction conditions (.alpha.)
associated with this 1:1 link. Similarly, T2 links F2 with R2 under
conditions .beta.. Transformations T3 and T4 are each unique
transformations despite being associated with a common fragment,
F3. Transformation T3 links common fragment F3 with reagent R3
under conditions .alpha., while transformation T4 links the common
fragment F3 with another reagent, R4, under the different
conditions, conditions .delta.. For example reagent R3 might be an
alkyl chloride while R4 might be an alkyl iodide. While these
reagents are similar (they are both alkyl halides), they might be
used under different reaction conditions. Use of different reagents
to effect the introduction of the same fragment into the compound
being generated in silico represents two unique transformations.
This indicates two distinct or unique synthetic ways of introducing
the same fragment into the compound. Depending upon the totality of
the chemical steps involved in synthesizing the compound, one
transformation may be preferred over other transformations that
introduce the same fragment into the compound.
[0193] Transformation T5 links fragment F4 with reagent R5. R5 is a
mixture of reagents, such as (R)- and (S)-stereoisomers, D- and
L-isomers, or two or more different reagents. As a result, use of
R5 leads to the introduction of a mixture of fragments F4 into the
compound. The art-skilled will recognize that the multiple reagents
in R5 are selected such that they are capable of being mixed
together, do not react with each other, and react under similar
reaction conditions. For example, R5 may be comprised of a mixture
of acid halides. These do not react with each other, but do react
similarly with a nucleophile under similar conditions. It is also
recognized by the art-skilled that a reagent is not limited to only
one or two components or constituent reagents, but in fact may
comprise of two, three, four, five or more reagents or
components.
[0194] When using a mixture of reagents, each of the individual
component reagents may have different chemical reactivity rates. If
a correction is not made for this, this could result in their
products being unequally represented in the product compounds. This
is solved by adjusting the concentration of each reagent in the
reaction mixture relative to the other reagents in the mixture such
that the relative rates are the same. This is effected by comparing
to the reactivity of each of the reagents to a chosen standard
reagent. The standardized reactivity rates can then be used to
adjust the concentration of each constituent reagent in the reagent
mixture to compensate for the varied reaction rates. Thus a mixture
of reagents with different reaction rates may be used in one
reagent mixture to still generate equivalent quantities of the
desired compounds in the library.
[0195] Transformations T6 and T7 are similar to transformations T3
and T4 except that conditions identifying each of these
transformations are different. Transformation T6 links fragment F5
with reagent R6 under conditions .epsilon., while transformation T7
links the same fragment F5 with a different reagent R7 under
different conditions (condition .alpha.). As the conditions
associated with transformations T6 and T7 are different, this
allows selection of compatible chemistries with other fragments
during any particular synthesis being used. This is a very useful
and very important consideration in actually synthesizing real
libraries. When it is desired to introduce fragment F5 into the
compound, the actual chemistries used to build the compound can be
initialy be considered in selecting transformation T6 or T7, and
thus reagents R6 or R7. This is in direct opposition to any
chemical database generator that only considers the compound
structure not the actual chemistries necessary to build a
compound.
[0196] Transformations T9 and T10 link fragment F7 with reagent R9
and fragment F8 with reagent R10, respectively. Both
transformations are identified to be associated with reaction
conditions .gamma.. Fragments F7 and F8 have three attachment
sites, but it is recognized that these fragments may have more than
three attachment sites, thereby increasing the complexity of the
compounds generated, and increasing the number of rounds that may
be employed to attach other fragments. For the three sites
illustrated, if three sets of different reagent mixtures each have
five reagents in the set are used, then 125 compounds will be
generated for fragment F7 and a further 125 compounds will be
generated for fragment F8.
[0197] The methods of the present invention may be used to generate
single compounds or mixtures of compounds. A mixture comprises two
or more compounds and may involve the use of two or more reagents
(thus introduction of two or more fragments) at the outset of
library generation, introduction of a mixture of reagents (thus a
mixture of fragments) at a subsequent stage of library generation,
or a combination of both such techniques. FIGS. 24 and 25
illustrate this aspect of the present invention.
[0198] As shown in FIG. 24, the methods of the present invention
may be used to generate single compounds such as C1 and C4, or may
also be used to generate a mixture of compounds, M1, comprising
compounds C2 and C3. Library generation commences with selecting
fragment F7 (with three attachment sites), in the first round (i.e.
round n). In the second synthesis round (i.e. round n+1), F7 is
combined with fragment F2, constituting synthetic pathway P1a, and
resulting in the formation of complex fragment CF1. F7 possesses
three attachment sites (i.e. X, Y and Z). Thus round n+1 will not
be complete until each of X, Y and Z have been used, if desired, to
attach other fragments to. Stepping around each of X, Y and Z, and
attaching fragments to these sites, occurs in that sequential
order. Once sites X, Y and Z of the fragment selected in the first
synthesis round (i.e. round n) have been exhausted, stepping around
the attachment sites present in the next added fragment constitutes
the next synthesis round (i.e. the third synthesis round, or round
n+2). Here again, when all desired attachment sites on this
fragment have been used, that particular synthesis round is
complete. This attachment iteration around the desired and
available attachment sites of the fragments added continues until
the desired compounds have been generated.
[0199] As shown in FIG. 24, CF1 is next subjected to synthetic
pathway P1b wherein fragment F1 is introduced into CF1, thereby
forming complex fragment CF2. CF2 is then subjected to synthetic
pathway P1c wherein fragment F5 is added to CF2, leading to the
formation of complex fragment CF3. This completes synthesis round
n+1 (i.e. the second round of fragment introduction, or synthesis,
to build the compound). As fragment F5 has two attachment sites,
CF3 has an available attachment site (i.e. site Y). Introduction of
fragments to this site (Y site) constitutes synthesis round n+2
(i.e. the third round) because all the desired attachment sites on
the previously added fragment have been exhausted. Next, CF3 is
subjected to synthetic pathway P2 wherein fragment F4 is introduced
into CF3 at attachment site Y. As F4 is a mixture of two
components, a mixture (M1) of two compounds, C2 and C3, is
generated.
[0200] A single compound, however, may also be generated using the
present scheme of fragment introduction. Thus, compound C1 can be
generated by subjecting CF3 to synthetic pathway P1d wherein CF3 is
combined with fragment F3, which attaches to site Y in CF3. The
introduction of fragment F3 into CF3 constitutes the third
synthesis round (i.e. round n+2), leading to the generation of
C1.
[0201] Alternately, CF3 can be subjected to synthetic pathway P3a
wherein fragment F6 is introduced into CF3 to form CF4. This
represents the third synthesis round (i.e. round n+2). CF4 has one
more available attachment site (i.e. site Y) to which fragment F2
may be attached via synthetic pathway P3b. This leads to the
generation of compound C4 which is a compound of increased
complexity because of the number of attachment sites on the chosen
fragments and synthetic pathways employed. The addition of fragment
F6 to CF4 constitutes the third synthesis round (i.e. round n+2).
Addition of fragment F2 to CF4 represents the fourth synthesis
round, or round n+3, because P3b involves addition of a fragment
(fragment F2) onto a site (i.e. site Y in CF4) which has been
generated by adding fragment F6 to CF3, thus exhausting the
available attachment sites on the previously added fragment in CF4
(i.e. fragment F5). That is, the addition of fragment F6 completed
round n+2 (or the third synthesis round) because F6 attached to the
last available attachment site on CF3 (i.e. site Y in CF3).
[0202] For the reactiosn effected at path P1c in FIG. 24, a single
fragment (F5) can be added to CF2 via use of either reagents R6 or
R7 (as thus via the transformations associated with R6 and R7).
While these additions are represented as two unique transformations
for the purpose of tracking in the database on the invention, these
additions in effect perform the same chemical conversion. Thus, the
simultaneous tracking of compounds generated according to the
methods of the invention is useful not only in working with virtual
libraries of compounds, but also provide the user with a choice of
synthetic pathways along which the compounds can be actually
synthesized. This tracking aspect of the present invention is,
therefore, a novel and unique way to account for the fragments
being introduced, the related transformations (or reactions)
associated with the fragments, and the alternate transformations
that lead to the introduction of a common fragment into the desired
compounds. The present invention allows not only the tracking of
individual compounds that are generated by the use of multiple
reagents, but also allows for the simultaneous tracking of multiple
compounds that are generated via multiple transformations. While
the methods described herein represent the tracking aspects of the
invention in terms of symbolic representations or tables, it is
recognized by the art-skilled that a variety of computer
algorithmic codes and techniques may be employed for the individual
or simultaneous tracking aspects described above.
[0203] The present invention further provides methods for the
one-pot generation of mixtures of compounds by commencing the
library generation using different starting fragments in a one-pot
fashion. One-pot generation or synthesis of compounds refers to the
formation of multiple compounds in a single reaction vessel (i.e.
one pot). This is possible if compatible chemistries are selected.
Examples of such single vessels include but are not limited to
multiple well plates, e.g. a 96-well plate, reactions flasks, e.g.
a 25 M1 flask, or even an industrial reactor. The reactions, or
transformations, are performed in one vessel reagrdless of the size
of the reaction vessel. The concept of one-pot synthesis is
irrelevant to the generation of virtual libraries of compounds as
these virtual libraries are merely generated in silico. The concept
of one-pot synthesis becomes relevant, however, when the actual
synthesis of libraries of compounds is to be undertaken. Thus the
compounds can be tracked separately for compound building in order
to generate distinct chemical structures, however, they can be
group together for synthesis allowing them to be made in the same
"pot."
[0204] An example of a one-pot synthesis was shown in FIG. 24 with
the addition of the complex reagent R5 to form mixture M1. A
further one-pot synthesis is shown in FIG. 25, where a further
mixture of compounds is generated. Mixture M2 comprising compounds
C1 and C5 can be generated by starting with fragments F7 and F8 in
the first synthesis round (i.e. round n). Each of these fragments
have three attachment sites onto which other fragments can be
introduced. As a result, subjecting the two fragments to synthetic
pathway P1a wherein F7 and F8 are combined with fragment F5 at site
X, results in the one-pot formation of complex fragments CF1 and
CF5. CF1 and CF5 are next subjected to synthetic pathway P1b
wherein fragment F1 is introduced into CF1 and CF5 at site Y,
thereby forming complex fragments CF2 and CF6. CF2 and CF6 are next
subjected to synthetic pathway P1c wherein fragment F5 is
introduced into these complex fragments at site Z, forming CF3 and
CF7. This completes the second synthetic round (i.e. round n+1). As
fragment F5 contains two attachment sites, after introduction into
CF3 and CF7, there is still available an attachment site (i.e. site
Y) for further introduction of another fragment. Thus CF3 and CF7
are converted to a mixture (M2) of compounds C1 and C5 via
synthetic pathway P1d wherein CF3 and CF7 are combined with
fragment F3 which attaches to the Y site on fragment F5 in CF3 and
CF7. The introduction of fragment F3 at site Y in CF3 and CF7
represents the third synthetic round (i.e. round n+2).
[0205] Yet another symbolic example of the one-pot generation of
mixtures of compounds, in accordance with the present invention, is
shown in FIG. 26. In silico generation of compounds commences with
the selection of fragment F7, which has three sites of attachment
(X, Y, and Z). This represents the first synthesis round (i.e.
round n). Next, F7 is subjected to synthetic pathway P1a wherein F7
is combined with fragment F2. F2 attaches to site X on fragment F7,
forming complex fragment CF1. At this stage, CF1 is subjected to
two synthetic pathways, P1b and P1b'. P1b employs fragment F1 which
is introduced onto site Y on CF1, thereby forming complex fragment
CF2, while P1b' employs fragment F3 which is introduced onto site Y
on CF1, thereby forming complex fragment CF8. Thus a mixture of
complex fragments (CF2 and CF8) are formed. Both fragments, F1 and
F3 can be introduced together (such as from a single reagent bottle
when actual synthesis is being undertaken) for the one-pot
generation of compounds if the chemistries associated with
introduction of these fragments into the compounds are compatible.
If not, these fragments can be introduced separately. Next, CF2 and
CF8 are subjected to synthetic pathway P1c wherein both complex
fragments are combined with fragment F5 which attaches to site Z on
CF2 and CF8, thereby forming complex fragments CF3 and CF9. The
formation of CF3 and CF9 completes the second synthesis round (i.e.
round n+1). As fragment F5 has two sites of attachment, site Y is
still available for attachment to another fragment. Therefore, CF3
is subjected to synthetic pathway P3 wherein CF3 is combined with
fragment F4. Introduction of F4 represents the third synthesis
round (i.e. round n+2). F4 is a mixture of fragments (and
introduced by adding a mixture of reagents), as shown in FIG. 22.
As a result, synthetic pathway P2 leads to the generation of
compounds C2 and C3. Simultaneously, CF9 combines with fragment F4,
via synthetic pathway P2', leading to the generation of compounds
C7 and C8. Thus mixture M3 is formed comprising compounds C2, C3,
C7 and C8.
[0206] The present invention also provides methods for the
generation of increasingly complex mixtures of compounds. An
example is shown in FIGS. 27a and 27b where mixture M4 is generated
and comprises sixteen compounds. The compounds in mixture M4 can be
generated by starting with fragments F7 and F8 in the first
synthesis round (i.e. round n). These fragments can then be
combined with fragment F2, which is introduced at site X in each of
F7 and F8, forming complex fragment CF1 and CF5. Following this, a
mixture of fragments F1 and F3 are introduced into CF1 and CF5 at
site Y of these complex fragments, leading to the formation of four
complex fragments, CF2, CF6, CF8 and CF11. These complex fragments
are next combined with a mixture of fragments F5 and F6. Both F5
and F6 have two attachment sites such that site X on F5 and F6
attaches to site Z on CF2, CF6, CF8 and CF11 forming a mixture of
eight complex fragments, CF3, CF7, CF9, CF12, CF13, CF14, CF15 and
CF16. This completes the second synthesis round (i.e. round n+1).
As fragments F5 and F6 have two attachment sites, X and Y, the
abovementioned eight complex fragments have one more available
attachment site (i.e. site Y) onto which another fragment may be
introduced. Attachment of a fragment to site Y on these eight
complex fragments represents the third synthesis round (i.e. round
n+2). Next, fragment F4 is introduced into CF3, CF7, CF9, CF12,
CF13, CF14, CF15 and CF16. As fragment F4 is a mixture of two
constituent fragments, sixteen compounds are generated: C2, C3, C7,
C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19 and C20.
Thus it can be seen that by using multiple fragments in a one-pot
fashion and combining with mixtures of fragments, mixtures of
compounds of increasing complexity can be generated. The example in
FIGS. 27a and 27b shows sixteen unique compounds being generated as
mixture M4 when the library is generated by starting with two
fragments. It is recognized by the art-skilled that if the library
generation is commenced with more than two fragments or multiple
fragments are added to the same precursor fragment, even more
complex mixtures of compounds can be generated.
[0207] The present invention also provides methods for keeping
track of fragment addition in the various synthesis rounds. This
system of accounting is accomplished by tabulation of the synthesis
rounds which are correlated with addition of fragments. While for
the purposes of illustration of the invention, a tabulation method
of tracking fragment addition is described herein, it will be
recognized by the art-skilled that other algorithms, algorithmic
codes, computer readable mediums and various software coding
techniques know to those skilled in the computer arts may be used
for such tracking. The tables tracking fragment addition can be
used to produce structural representations of compounds and create
virtual libraries where actual synthesis of the compounds is not
desired. Tables tracking transformations, however, can be used to
synthesize compounds by selecting the appropriate transformations,
and in the case of multiple transformations, selecting the
preferable transformations to introduce the required fragment into
the compounds being synthesized.
[0208] FIG. 28 is descriptive of compound C1 in terms of the
fragments added in each synthesis round. The first synthesis round
(i.e. round n) commences with the selection of fragment F7. This is
followed by the sequential addition of fragments F2, F1 and F5 in
the second synthesis round (i.e. round n+1). Finally, compound C1
is generated by the addition of fragment F3 in the third synthesis
round (i.e. round n+2). The compounds thus generated can be stored
as a 2-dimensional virtual library, or may be converted to a
3-dimensional virtual library that can be used for in silico
docking to desired target molecules.
[0209] For the generation of virtual libraries of compounds and for
docking the library members onto target molecules, it suffices to
add compounds to the relational database in terms of its fragments
to track the addition of fragments in the various synthetic rounds.
However, when the actual synthesis of desired compounds of a
library is to be undertaken, it becomes necessary to specify the
actual synthetic steps, reagents, solvents, concentrations,
auxiliary compounds needed and other various synthetic factors in
order to effect such an actual synthesis of real chemical
compounds. Such synthetic steps, reagents, solvents, concentrations
and auxiliary compounds are, in fact, incorporated in to the above
described transformations. Thus by employing the concept of
transformations, the present invention provides methods to track
the compounds generated not only in terms of the fragments added
but as well as the synthetic parameters necessary for each
synthesis round.
[0210] FIG. 28 also shows the generation of compound C1 in terms of
the various transformations employed in the synthesis rounds. Four
synthesis pathways lead to the synthesis of compound C1 because of
the availability of multiple transformations that can introduce the
same fragment into the compound being synthesized. Thus, as seen in
FIG. 28, selection of fragment F7 constitutes transformation T9 in
the first synthesis round (i.e. round n). This is followed by the
addition of fragment F2 which is achieved by employing
transformation T2. Next, fragment F1 is added via transformation
T1. Fragment F5, however, may be added by employing either reagent
R6 via transformation T6 along synthesis paths 1 and 3, or reagent
R7 via transformation T7 along synthesis paths 2 and 4. Similarly,
the final fragment F3 can be added by using either reagent R3 via
transformation T3 along synthesis paths 1 and 2, or reagent R4 via
transformation T4 along synthesis paths 3 and 4. Thus FIG. 28 shows
that compound C1 can be actually synthesized via one of four
different synthetic schemes which can be tracked or tabulated and
accounted for using the methods of the present invention. Each of
the four tables is completely descriptive of each of the four
synthetic pathways for the preparation of C1. Thus, a user of the
present invention has available all the alternate pathways of
performing the same reaction (i.e. introducing the same fragment),
and can select the preferable or most appropriate synthetic route
to preparing the desired compounds.
[0211] FIG. 29 shows a similar transformation tracking table for
compounds C2 and C3 in mixture M1. Synthesis of compounds C2 and C3
commences with selection of fragment F7 which represents
transformation T9 (step 1 in FIG. 29) in the first synthesis round
(i.e. round n). Next, F7 is combined with fragment F2 via
transformation T2 in the second synthesis round (i.e. round n+1)
(step 2). In the same round, fragment F1, via transformation T1,
and fragment F5, via transformation T7 are added sequentially
(steps 3 and 4). Finally, fragment F4 is added in the third
synthesis round (i.e. round n+2). As F4 is a mixture of two
constituent fragments (because of two constituent reagents), the
table is duplicated at this stage (step 5) to account for the
different synthetic ways in which transformation T5 may be
accomplished (i.e. T5.sup.1 and T5.sup.2). Step 5 represents
compounds C2 and C3. Thus it can be seen that, in accordance with
the present invention, whenever there is more than one reagents
associated with a particular transformation, the table is
duplicated as many times as there are such reagents.
[0212] FIG. 30 shows a transformation tracking table for compounds
C1 and C5 in mixture M3. As the synthesis commences with two
fragments, F7 and F8, tracking begins with two parallel tables
(step 1 in FIG. 30). In the first synthesis round (i.e. round n),
F7 is selected via transformation T9, while F8 is selected via
transformation T10. The second synthesis round (i.e. round n+1)
commences at step 2 with the introduction of fragment F2 via
transformation T2. In step 3, transformation T1 introduces fragment
F1 into the compound. In step 4, transformation T7 introduces
fragment F5. This completes the second synthesis round (i.e. round
n+1). Finally, in the third synthesis round (i.e. round n+2),
transformation T4 is used to introduce fragment F3 (at step 5)
producing mixture M2 comprising compounds C1 and C5. In this
example, the tables are duplicated early in the synthetic scheme
because of the use of a mixture of fragments F7 and F8 at the
outset.
[0213] The transformation tracking table for compounds C2, C3, C7
and C8 of mixture M3 are shown in FIG. 31. The synthesis of these
compounds commences with the first synthesis round (i.e. round n)
in which fragment F7 is selected. This represents transformation T9
(shown in step 1 in FIG. 31). Step 2 in FIG. 31 depicts the second
synthesis round (i.e. round n+1) and involves the addition of
fragment F2 via transformation T2. While steps 1 and 2 involve
single transformations each, step 3 involves two different
transformations because two different fragments are being
introduced into the compounds through the use of two different
reagents. Therefore, at step 3 the table is twice duplicated
because two different reagents are being employed to introduce two
different fragments via two different transformations. In step 3,
transformation T1 is used to introduce fragment F1 while
transformation T3 is used to introduce fragment F3. The second
synthesis round (i.e. round n+1) is completed at step 4 with
transformation T7 which introduces fragment F5. In the final
synthesis round (i.e. the third round or round n+2), transformation
T5 is used to introduce fragment F4. As F4 is a mixture of two
constituent fragments, each table at step 5 is twice duplicated for
transformations T5.sup.1 and T5.sup.2 which represent each of the
constituent fragments of F4.
[0214] These figures represent merely one manner in which the
various fragments, reagents and transformations may be tracked
during the generation or synthesis of single compounds or mixtures
of compounds. It will, however, be recognized by the art-skilled
that various other algorithm schemes may be employed to track and
account for the fragments being introduced via transformations when
compounds are being generated in silico.
[0215] The library members or compounds generated according to the
methods of the present invention can be converted into
three-dimensional representations using commercially available
software. Next, the compounds, in their three-dimensional
structures can be docked onto identified targets, also represented
as three-dimensional structures.
[0216] Docking of these library members (or ligands) entails the in
silico binding of the members to desired target molecules. A
variety of theoretical and computational methods are known in the
literature to study and optimize the interactions of small
molecules with biological targets such as proteins and nucleic
acids. These structure-based drug design tools have been very
useful in modeling the interactions of proteins with small molecule
ligands and in optimizing these interactions. Typically this type
of study was performed when the structure of the protein receptor
was known by querying individual small molecules, one at a time,
against this receptor. Usually these small molecules had either
been co-crystallized with the receptor, were related to other
molecules that had been co-crystallized or were molecules for which
some body of knowledge existed concerning their interactions with
the receptor. A significant advance in this area was the
development of a software program called DOCK that allows
structure-based database searches to find and identify the
interactions of known molecules to a receptor of interest (Kuntz et
al., Acc. Chem. Res., 1994, 27, 117; Gschwend and Kuntz, J.
Compt.-Aided Mol. Des., 1996, 10, 123). DOCK allows the screening
of molecules, whose 3D structures have been generated in silico,
but for which no prior knowledge of interactions with the receptor
is available. DOCK, therefore, provides a tool to assist in
discovering new ligands to a receptor of interest. DOCK can thus be
used for docking the compounds prepared according to the methods of
the present invention to desired target molecules.
[0217] The DOCK program has been applied to protein targets and the
identification of ligands that bind to them. The DOCK software
program consists of several modules, including SPHGEN (Kuntz et
al., J. Mol. Biol., 1982, 161, 269) and CHEMGRID (Meng et al., J.
Comput. Chem., 1992, 13, 505). SPHGEN generates clusters of
overlapping spheres that describe the solvent-accessible surface of
the binding pocket within the target receptor. Each cluster
represents a possible binding site for small molecules. CHEMGRID
precalculates and stores in a grid file the information necessary
for force field scoring of the interactions between binding
molecule and target. The scoring function approximates molecular
mechanics interaction energies and consists of van der Waals and
electrostatic components. DOCK uses the selected cluster of spheres
to orient ligands molecules in the targeted site on the receptor.
Each molecule within a previously generated 3D database is tested
in thousands of orientations within the site, and each orientation
is evaluated by the scoring function. Only that orientation with
the best score for each compound so screened is stored in the
output file. Finally, all compounds of the database are ranked in
order of their scores and a collection of the best candidates may
then be screened experimentally.
[0218] Using DOCK, ligands have been identified for certain protein
targets. Recent efforts in this area have resulted in reports of
the use of DOCK to identify and design small molecule ligands that
exhibit binding specificity for nucleic acids such as RNA double
helices. While RNA plays a significant role in many diseases such
as AIDS, viral and bacterial infections, few studies have been made
on small molecules capable of specific RNA binding. Compounds
possessing specificity for the RNA double helix, based on the
unique geometry of its deep major groove, were identified using the
DOCK methodology (Chen et al., Biochemistry, 1997, 36, 11402; Kuntz
et al., Acc. Chem. Res., 1994, 27, 117). Using a recent X-ray
structure for r(UAAGGAGGUGAU).r(AUCACCUCCUUA) as the model
structure for the A-form RNA duplex, DOCK identified several
aminoglycosides as candidate ligands, characterized by shape
complementarity to the RNA groove. Binding experiments then
revealed that one of these aminoglycosides not only bound
preferentially to RNA over B-form DNA but also that the ligand
binds in the targeted RNA major groove. Recently, the application
of DOCK to the problem of ligand recognition in DNA quadruplexes
has also been reported (Chen et al., Proc. Natl. Acad. Sci., 1996,
93, 2635).
[0219] As yet there has been no report of the evaluation of virtual
libraries against RNA targets. Certain reports of the generation of
virtual libraries are available from the standpoint of library
design, generation, and screening against protein targets.
Likewise, some efforts in the area of generating RNA models have
been reported in the literature. However, there are no reports on
the use of structure-based design approaches to query virtual
libraries against three-dimensional models of RNA structure so as
to identify ligands, such as small molecules, oligonucleotides or
other nucleic acids, that bind to such targets. The present
invention provides a solution to this problem by allowing the
building of three-dimensional models of RNA structure, the building
of virtual libraries of ligands, including small molecules,
polymeric compounds, oligonucleotides and other nucleic acids,
screening of such virtual libraries against RNA targets in silico,
scoring and identifying the best potential binders from such
libraries, and finally, synthesizing such molecules in a
combinatorial fashion and testing them experimentally to identify
new ligands for such targets.
[0220] The methods of the present invention aid in the drug
discovery process by allowing the identification of those library
members which bind with high affinity to the target molecules and,
therefore, represent molecules that may be actually synthesized and
developed as lead drug candidates.
[0221] The present invention is also directed to computational
methods employed for the in silico design and synthesis of
combinatorial libraries of small molecules. The library members are
generated in silico. The present invention also encompasses methods
for tracking and storing the information generated during the in
silico creation of library members into relational databases for
later access and use of this information to synthesize chemical
compounds corresponding to those genereated in silico. For the
purposes of this specification, in silico refers to the creation in
a computer memory, i.e., on a silicon or other like chip. Stated
otherwise in silico means "virtual."
[0222] According to the methods of the present invention, each
compound or library member is dissected into its component or
constituent parts referred to as fragments. Thus each compound that
is generated is considered to be comprised of constituent fragments
such that the sum of the molecular formulas of each of the
fragments when added together totals the molecular formula of the
compound generated. This dissection can be done in a variety of
ways using chemical intuition. Thus a variety of components of
fragments may be identified, each of which lend themselves to
readily available reagents or reactions to generate diverse
compounds. Further, each fragment is associated with at least one
reagent, which represents the necessary chemical to be used to
introduce that desired fragment into the compound being generated
in silico. Dissection of compounds is based on the ease of
synthesis of the reagents, commercial availability of the reagents,
or a combination of both. Each of the fragments and reagents are
stored in a relational database and are described in terms of
identifying characteristics in the database. A fragment may be
available from a variety of starting materials or reaction schemes.
So when a library is being generated, which entails building a
database, the fragments used in building that library can be stored
in the database using the corresponding set of reagents and
reaction conditions. When another library is to be generated, the
fragment information stored in the database is now available for
use in the generation of the new library of compounds. Similarly,
when a third library is being generated, an even greater quantity
of fragment, reagent, and reaction information is available in the
database. Thus the methods of the present invention represent a
dynamic method of building a database associated with building
libraries of compounds. Initial library generation requires
database input for fragments, reagents and transformations
necessary for desired library. As the database grows, however, an
increasing number of fragments and reagents are available in the
database, which simplifies the generation of subsequent libraries
of compounds and makes for more routine combinatorial synthetic
efforts which can be accomplished with increasing ease and
efficacy.
[0223] Fragments that are recorded in the database may be defined
using identifying characteristics. Identifying characteristics
defining fragments include a structural representation (as a
2-dimensional or 3-dimensional file), name, molecular weight,
molecular formula, and attachment points or nodes (which denote
sites of attachment or linkage of the fragment to other fragments
of the compound being generated in silico). For the purpose of
describing this invention, 2-dimensional representations are used,
which are further simplified by the use of symbolic representations
without reference to any particular chemical entities. The symbolic
representations as used herein merely shows how fragments can be
tracked to further the methods of the present invention. Other
identifying characteristics may also be added to the database. Any
characteristic that is desired to be tracked may be included in the
database, including biological data, chemical reactivity rates, or
other physical or chemical properties. Further, a fragment may also
be created by modifying a reagent, and such modifications can be
added to the database in terms of changes made to the reagent
structure. Some of the identifying characteristics associated with
any fragment may be common to those of the corresponding reagent.
The related fragment thus created can then be stored in the
relational database.
[0224] Identifying characteristics defining reagents include a
structural representation, name, molecular weight, molecular
formula, and source, such as a commercial source or a unique
compound defined by the user. In case of a commercial source for
the reagent, a catalog number or a link to a web page can be
provided. Some commonalities may exist between the identifying
characteristics associated with a reagent and those associated with
the related fragment.
[0225] Further, in accordance with the present invention, a
compound is the sum of various transformations. Transformation is
the nomenclature attributed according to the present invention to a
chemical synthesis. A transformation is a 1:1 link between a
fragment and a reagent. Thus each transformation describes a unique
conversion of a reagent into the corresponding fragment as
introduced into a compound. When the compound being generated in
silico is broken down into its component fragments, and the
corresponding reagents have been identified, each fragment is
linked to the corresponding reagent in a 1:1 relationship in order
to describe a transformation. Thus, according to the present
invention, a transformation may be viewed as the source of a
fragment, thereby linking that fragment to a particular synthetic
method or reaction. This description of a transformation according
to the methods of the present invention also includes any auxiliary
reagents or conditions used to effect the reaction denoted by the
transformation, such as temperature and pressure requirements,
catalysts, activators, solvents, or other additives.
[0226] Each combination of a fragment and reagent in a 1:1 link
comprises a different transformation. Therefore, each
transformation is unique. The present invention allows the tracking
of fragments in terms of the reaction or transformation in which
those fragments are introduced into the compounds of the library.
Thus the database describes not only the compounds generated in
terms of their constituent fragments, but also in terms of the
synthetic pathways to produce those compounds, i.e. the related
transformations to generate the library compounds. In this manner,
a user of the present invention can generate a virtual library of
compounds by simply selecting the fragments desired. Alternately, a
user can also generate the compounds by selecting the chemical
pathways required for actual synthesis of the compounds. This is
accomplished by selecting the appropriate transformation associated
with the generation of the desired compounds. Here, the user uses
intuition or an in silico expert system to assist in selecting
those transformations that are expected to allow generation or
synthesis of the desired compounds. Each of the transformations
created in silico is stored in the relational database and
described in terms of identifying characteristics. Identifying
characteristics defining transformations include the fragment, the
reagent, and any auxiliary reagent or conditions necessary to
effect the conversion of the reagent into the fragment as
incorporated into the compound.
[0227] For example, consider in FIG. 14 the in silico generation of
compound CI according to the methods of the present invention. As
shown in FIG. 14, upon dissection of CI (molecular formula of
C.sub.12H.sub.18N.sub.2O.sub.5S.sub.1), its constituent fragments
can be denoted as F.sub.i (molecular formula of H.sub.2NO),
F.sub.ii (molecular formula of C.sub.5H.sub.9NO), and F.sub.iii
(molecular formula of C.sub.7H.sub.7O.sub.3S). F.sub.i can also be
a hydroxyl amine moiety linked to a solid support, i.e. P--O--NH,
wherein P is a solid support. The sum of the molecular formulas of
each of the fragments totals the molecular formula of compound
CI.
[0228] As shown in FIG. 15, each of the fragments, F.sub.i,
F.sub.ii, and F.sub.iii, are stored in a relational database, and
are described in terms of identifying characteristics including a
structural representation (which may be 2-dimensional or
3-dimensional), an identifier or name, molecular formula and
attachment points or nodes which signify sites on the fragment
which are linked to other fragments in compound CI. Other
information such as molecular weight can also be associated with
the fragment in the database.
[0229] As shown in FIG. 16, each of the corresponding reagents
(R.sub.i, R.sub.ii, and R.sub.iii) are also stored in the
relational database, and described in terms of identifying
characteristics. Identifying characteristics used to define the
reagents include a structural representation, and identifier or
name and molecular formula. As with the fragment, other associated
information such as molecular weight and source (such as a
commercial source verses user-supplied, amount on hand, special
handling, etc.) can also be stored in database in association with
the individual reagents.
[0230] Next, each of the transformations associated with the in
silico generation of compound CI are also stored in the relational
database. As shown in FIG. 17, transformation T.sub.i links reagent
R.sub.i with fragment F.sub.i, T.sub.ii links R.sub.ii with
F.sub.ii, and T.sub.iii links R.sub.iii with F.sub.iii in a 1:1
relationship. Also, associated with each transformation is the
necessary reaction condition, so that transformation T.sub.i is
associated with reaction condition alpha, T.sub.ii with reaction
condition beta, and T.sub.iii with reaction condition gamma. In the
case of transformation T.sub.iii, reagent R.sub.iii may be a
hydroxyl amine attached to a solid support so that fragment
F.sub.iii can be represented as a hydroxyl amine moiety attached to
a solid support.
[0231] While each fragment may be arrived at or generated by a
unique corresponding reagent, the present invention also
encompasses common fragments that may be generated via two or more
reagents, so that two or more transformations can lead to the same
fragment. As shown in FIG. 18, the common fragment
CH.sub.3--CH.sub.2--C(.dbd.O)-- may be arrived at via
transformation A, which employs reagent X (an acid chloride),
CH.sub.3--CH.sub.2--C(.dbd.O)Cl. The common fragment can also be
introduced into a compound being generated in silico via
transformation B, which employs reagent Y (an acid anhydride),
CH.sub.3--CH.sub.2--C(.db- d.O)--O--C(.dbd.O)--CH.sub.2--CH.sub.3.
Therefore, in accordance with the methods of the present invention,
a common fragment can be introduced into the compound via two or
more different reagents, and thus via two or more distinct
transformations.
[0232] Alternately, a common reagent may be employed to effect two
or more conversions forming two or more different fragments. This
then represents two or more different transformations associated
with different conditions. For example, as shown in FIG. 19a,
common reagent Z, CH.sub.3--CH.sub.2--NH.sub.2, can be employed to
introduce an alkene fragment into the compound under conditions
favoring Schiff's base formation. This represents transformation X.
The same common reagent Z, however, can also be employed to
introduce an amide fragment into the compound by using a different
set of conditions, constituting transformation Y. Thus, a common
reagent can introduce two or more different fragments into final
compounds being generated in silico, and can be associated with two
or more transformations depending upon the conditions associated
with each of those transformations.
[0233] Additionally, once a fragment has been introduced into a
compound, it can be further modified and converted into yet another
fragment without effecting any other chemical changes within the
compound formed. As an example, shown in FIG. 19b, consider common
reagent Z', CH.sub.3--CH.sub.2--C(.dbd.O)CH.sub.2--Cl. Common
reagent Z' corresponds to a fragment having the structure
CH.sub.3--CH.sub.2--C(.dbd.O)CH.sub.2-- -. Common reagent Z' may be
used to introduce an alkene fragment into the final compound,
representing transformation X', under conditions favoring reduction
and dehydration. Common reagent Z', however, can also be used to
introduce a hydroxyalkyl fragment into the final compound under
conditions favoring reduction. This represents transformation
Y'.
[0234] The present invention may be described more generally, in
terms of symbolic representations. Symbolic representations are
used to describe the methods of the present invention because such
representations are not limited to any particular chemistry.
Symbolic representations merely denote the manner of using the
present invention with multiple chemical entities. Each symbol used
in the representations describing the present invention may
represent one compound or multiple compounds because the present
invention is not limited to tracking a single compound, but may be
used to track a vast variety of compounds that can be
generated.
[0235] FIG. 20 shows the symbolic addition of fragments which
yields compound CI'. The fragments have structures F.sub.i,
F.sub.ii, and F.sub.iii that are added sequentially to yield
compound CI'. Structures F.sub.i', F.sub.ii', and F.sub.iii' are
symbolic representations of the fragments that constitute compound
CI'. These fragments can be stored in the relational database with
the corresponding identifying characteristics for each of them,
including the structural representation, name, molecular formula,
and attachment sites or nodes. A visual inspection of compounds C1
and C1' revels the commonality between the chemical compound C1 and
the symbolic representation of a compound C1' as well as the
chemical structure of the fragments and the symbolic structure of
the fragments.
[0236] A symbolic reagent table is shown in FIG. 21. Reagents R1 to
R10 can be described in terms of their structure, name, molecular
formula, molecular weight, and source as well as other information
that might be desired to be associated with the reagents. R3 and R4
are two different reagents, but may be used to introduce the same
fragment into a compound. This depends upon the reaction conditions
used as reagent R3 is used in a transformation associated with one
set of conditions, while reagent R4 is used in another
transformation associated with a different set of conditions. Also,
reagent R5 is comprised of a mixture of two reagents or components.
These may be (R)- and (S)-stereoisomers, D- and L-isomers, or may
be two completely different reagents. While R5 here is represented
as a mixture of only two reagents or components, it will be
recognized by the art-skilled that the methods of the present
invention may be practiced using a mixture of two or more reagents.
Typical reagent mixtures used in constructing libraries might have
four, five or more individual reagent constituting the mixture.
[0237] FIG. 22 shows a symbolic fragment table. Fragments F1 to F8
are stored in the relational database with identifying
characteristics that include a structural representation, name,
molecular weight, molecular formula, and attachment sites or nodes.
This table depicts symbolic representations of the various
fragments that are introduced into the compounds of the library by
the use of reagents symbolized in FIG. 21. Thus it can be seen that
fragment F1 can be introduced into the compound by employing
reagent R1. In fragment F1, X is an identifier for an attachment
site. This indicates that X is the site at which F1 attaches to
another fragment in a compound. Similarly, fragment F2 may be
introduced into a compound (attaching at its X site) by employing
reagent R2.
[0238] Fragment F3, however, can be introduced into the compound by
the use of either reagent R3 or R4. This allows for selection in
the choice of the reagent used, and also allows for the
consideration of the compatibility of the chemistries involved in
the introduction of other fragments into the compound. Next,
fragment F4 (which is a mixture of fragments) can be introduced via
the use of reagent R5, which is a mixture of reagents, as shown in
FIG. 21.
[0239] Fragment F5 has two attachment sites, indicating that other
fragments can attach at sites X and Y when F5 has been incorporated
into a compound. The presence of two attachment sites indicates
that two attachments may be undertaken to build a compound when
dealing with F5. Here again, as before, F5 can be introduced into
the compound using either of reagents R6 or R7, depending upon the
reaction conditions used and the chemistries involved when
introducing other fragments to build the compound.
[0240] Fragments F7 and F8 can be introduced into a compound being
created in silico by employing reagents R9 and R10, respectively.
Both these fragments have three attachment sites, indicating that
three attachments to other fragments can occur when using these
fragments to build a compound in silico. While fragments F7 and F8
have three attachment sites, it is recognized by the art-skilled
that more than three attachment sites may be present in a fragment,
allowing for more attachments to the fragment upon introduction
into a compound (with the use of an appropriate reagent).
[0241] With the fragment and reagent tables in place in the
relational database, a transformation table is created in
accordance with the methods of the present invention, by linking a
fragment with a reagent to form a unique transformation. FIG. 23
shows a symbolic transformation table where a fragment is linked to
a reagent in a 1:1 relationship. The identifying characteristics
describing each transformation include a 1:1 link (a one to one
link) between a fragment and a reagent, and the reaction conditions
which include, solvent, concentration, temperature and pressure
requirements, or auxiliary reagents necessary to effect the
introduction of the fragment into the compound by using an
appropriate reagent. Auxiliary reagents include catalysts,
activators, acids, bases or other chemicals or additives necessary
to effect the fragment introduction described. For example a base
can always be added with an alkyl halide to scavenge the acid
generated with use of the alkyl halide.
[0242] As seen in FIG. 23, transformation T1 links fragment F1 with
reagent R1. T1 also specifies the reaction conditions (.alpha.)
associated with this 1:1 link. Similarly, T2 links F2 with R2 under
conditions .beta.. Transformations T3 and T4 are each unique
transformations despite being associated with a common fragment,
F3. Transformation T3 links common fragment F3 with reagent R3
under conditions .alpha., while transformation T4 links the common
fragment F3 with another reagent, R4, under the different
conditions, conditions .delta.. For example reagent R3 might be an
alkyl chloride while R4 might be an alkyl iodide. While these
reagents are similar (they are both alkyl halides), they might be
used under different reaction conditions. Use of different reagents
to effect the introduction of the same fragment into the compound
being generated in silico represents two unique transformations.
This indicates two distinct or unique synthetic ways of introducing
the same fragment into the compound. Depending upon the totality of
the chemical steps involved in synthesizing the compound, one
transformation may be preferred over other transformations that
introduce the same fragment into the compound.
[0243] Transformation T5 links fragment F4 with reagent R5. R5 is a
mixture of reagents, such as (R)- and (S)-stereoisomers, D- and
L-isomers, or two or more different reagents. As a result, use of
R5 leads to the introduction of a mixture of fragments F4 into the
compound. The art-skilled will recognize that the multiple reagents
in R5 are selected such that they are capable of being mixed
together, do not react with each other, and react under similar
reaction conditions. For example, R5 may be comprised of a mixture
of acid halides. These do not react with each other, but do react
similarly with a nucleophile under similar conditions. It is also
recognized by the art-skilled that a reagent is not limited to only
one or two components or constituent reagents, but in fact may
comprise of two, three, four, five or more reagents or
components.
[0244] When using a mixture of reagents, each of the individual
component reagents may have different chemical reactivity rates. If
a correction is not made for this, this could result in their
products being unequally represented in the product compounds. This
is solved by adjusting the concentration of each reagent in the
reaction mixture relative to the other reagents in the mixture such
that the relative rates are the same. This is effected by comparing
to the reactivity of each of the reagents to a chosen standard
reagent. The standardized reactivity rates can then be used to
adjust the concentration of each constituent reagent in the reagent
mixture to compensate for the varied reaction rates. Thus a mixture
of reagents with different reaction rates may be used in one
reagent mixture to still generate equivalent quantities of the
desired compounds in the library.
[0245] Transformations T6 and T7 are similar to transformations T3
and T4 except that conditions identifying each of these
transformations are different. Transformation T6 links fragment F5
with reagent R6 under conditions .epsilon., while transformation T7
links the same fragment F5 with a different reagent R7 under
different conditions (condition a). As the conditions associated
with transformations T6 and T7 are different, this allows selection
of compatible chemistries with other fragments during any
particular synthesis being used. This is a very useful and very
important consideration in actually synthesizing real libraries.
When it is desired to introduce fragment F5 into the compound, the
actual chemistries used to build the compound can be initialy be
considered in selecting transformation T6 or T7, and thus reagents
R6 or R7. This is in direct opposition to any chemical database
generator that only considers the compound structure not the actual
chemistries necessary to build a compound.
[0246] Transformations T9 and T10 link fragment F7 with reagent R9
and fragment F8 with reagent R10, respectively. Both
transformations are identified to be associated with reaction
conditions .gamma.. Fragments F7 and F8 have three attachment
sites, but it is recognized that these fragments may have more than
three attachment sites, thereby increasing the complexity of the
compounds generated, and increasing the number of rounds that may
be employed to attach other fragments. For the three sites
illustrated, if three sets of different reagent mixtures each have
five reagents in the set are used, then 125 compounds will be
generated for fragment F7 and a further 125 compounds will be
generated for fragment F8.
[0247] The methods of the present invention may be used to generate
single compounds or mixtures of compounds. A mixture comprises two
or more compounds and may involve the use of two or more reagents
(thus introduction of two or more fragments) at the outset of
library generation, introduction of a mixture of reagents (thus a
mixture of fragments) at a subsequent stage of library generation,
or a combination of both such techniques. FIGS. 24 and 25
illustrate this aspect of the present invention.
[0248] As shown in FIG. 24, the methods of the present invention
may be used to generate single compounds such as C1 and C4, or may
also be used to generate a mixture of compounds, M1, comprising
compounds C2 and C3. Library generation commences with selecting
fragment F7 (with three attachment sites), in the first round (i.e.
round n). In the second synthesis round (i.e. round n+1), F7 is
combined with fragment F2, constituting synthetic pathway P1a, and
resulting in the formation of complex fragment CF1. F7 possesses
three attachment sites (i.e. X, Y and Z). Thus round n+1 will not
be complete until each of X, Y and Z have been used, if desired, to
attach other fragments to. Stepping around each of X, Y and Z, and
attaching fragments to these sites, occurs in that sequential
order. Once sites X, Y and Z of the fragment selected in the first
synthesis round (i.e. round n) have been exhausted, stepping around
the attachment sites present in the next added fragment constitutes
the next synthesis round (i.e. the third synthesis round, or round
n+2). Here again, when all desired attachment sites on this
fragment have been used, that particular synthesis round is
complete. This attachment iteration around the desired and
available attachment sites of the fragments added continues until
the desired compounds have been generated.
[0249] As shown in FIG. 24, CF1 is next subjected to synthetic
pathway P1b wherein fragment F1 is introduced into CF1, thereby
forming complex fragment CF2. CF2 is then subjected to synthetic
pathway P1c wherein fragment F5 is added to CF2, leading to the
formation of complex fragment CF3. This completes synthesis round
n+1 (i.e. the second round of fragment introduction, or synthesis,
to build the compound). As fragment F5 has two attachment sites,
CF3 has an available attachment site (i.e. site Y). Introduction of
fragments to this site (Y site) constitutes synthesis round n+2
(i.e. the third round) because all the desired attachment sites on
the previously added fragment have been exhausted. Next, CF3 is
subjected to synthetic pathway P2 wherein fragment F4 is introduced
into CF3 at attachment site Y. As F4 is a mixture of two
components, a mixture (M1) of two compounds, C2 and C3, is
generated.
[0250] A single compound, however, may also be generated using the
present scheme of fragment introduction. Thus, compound C1 can be
generated by subjecting CF3 to synthetic pathway P1d wherein CF3 is
combined with fragment F3, which attaches to site Y in CF3. The
introduction of fragment F3 into CF3 constitutes the third
synthesis round (i.e. round n+2), leading to the generation of
C1.
[0251] Alternately, CF3 can be subjected to synthetic pathway P3a
wherein fragment F6 is introduced into CF3 to form CF4. This
represents the third synthesis round (i.e. round n+2). CF4 has one
more available attachment site (i.e. site Y) to which fragment F2
may be attached via synthetic pathway P3b. This leads to the
generation of compound C4 which is a compound of increased
complexity because of the number of attachment sites on the chosen
fragments and synthetic pathways employed. The addition of fragment
F6 to CF4 constitutes the third synthesis round (i.e. round n+2).
Addition of fragment F2 to CF4 represents the fourth synthesis
round, or round n+3, because P3b involves addition of a fragment
(fragment F2) onto a site (i.e. site Y in CF4) which has been
generated by adding fragment F6 to CF3, thus exhausting the
available attachment sites on the previously added fragment in CF4
(i.e. fragment F5). That is, the addition of fragment F6 completed
round n+2 (or the third synthesis round) because F6 attached to the
last available attachment site on CF3 (i.e. site Y in CF3).
[0252] For the reactiosn effected at path P1c in FIG. 24, a single
fragment (F5) can be added to CF2 via use of either reagents R6 or
R7 (as thus via the transformations associated with R6 and R7).
While these additions are represented as two unique transformations
for the purpose of tracking in the database on the invention, these
additions in effect perform the same chemical conversion. Thus, the
simultaneous tracking of compounds generated according to the
methods of the invention is useful not only in working with virtual
libraries of compounds, but also provide the user with a choice of
synthetic pathways along which the compounds can be actually
synthesized. This tracking aspect of the present invention is,
therefore, a novel and unique way to account for the fragments
being introduced, the related transformations (or reactions)
associated with the fragments, and the alternate transformations
that lead to the introduction of a common fragment into the desired
compounds. The present invention allows not only the tracking of
individual compounds that are generated by the use of multiple
reagents, but also allows for the simultaneous tracking of multiple
compounds that are generated via multiple transformations. While
the methods described herein represent the tracking aspects of the
invention in terms of symbolic representations or tables, it is
recognized by the art-skilled that a variety of computer
algorithmic codes and techniques may be employed for the individual
or simultaneous tracking aspects described above.
[0253] The present invention further provides methods for the
one-pot generation of mixtures of compounds by commencing the
library generation using different starting fragments in a one-pot
fashion. One-pot generation or synthesis of compounds refers to the
formation of multiple compounds in a single reaction vessel (i.e.
one pot). This is possible if compatible chemistries are selected.
Examples of such single vessels include but are not limited to
multiple well plates, e.g. a 96-well plate, reactions flasks, e.g.
a 25 mL flask, or even an industrial reactor. The reactions, or
transformations, are performed in one vessel reagrdless of the size
of the reaction vessel. The concept of one-pot synthesis is
irrelevant to the generation of virtual libraries of compounds as
these virtual libraries are merely generated in silico. The concept
of one-pot synthesis becomes relevant, however, when the actual
synthesis of libraries of compounds is to be undertaken. Thus the
compounds can be tracked separately for compound building in order
to generate distinct chemical structures, however, they can be
group together for synthesis allowing them to be made in the same
"pot."
[0254] An example of a one-pot synthesis was shown in FIG. 24 with
the addition of the complex reagent R5 to form mixture M1. A
further one-pot synthesis is shown in FIG. 25, where a further
mixture of compounds is generated. Mixture M2 comprising compounds
C1 and C5 can be generated by starting with fragments F7 and F8 in
the first synthesis round (i.e. round n). Each of these fragments
have three attachment sites onto which other fragments can be
introduced. As a result, subjecting the two fragments to synthetic
pathway P1a wherein F7 and F8 are combined with fragment F5 at site
X, results in the one-pot formation of complex fragments CF1 and
CF5. CF1 and CF5 are next subjected to synthetic pathway P1b
wherein fragment F1 is introduced into CF1 and CF5 at site Y,
thereby forming complex fragments CF2 and CF6. CF2 and CF6 are next
subjected to synthetic pathway P1c wherein fragment F5 is
introduced into these complex fragments at site Z, forming CF3 and
CF7. This completes the second synthetic round (i.e. round n+1). As
fragment F5 contains two attachment sites, after introduction into
CF3 and CF7, there is still available an attachment site (i.e. site
Y) for further introduction of another fragment. Thus CF3 and CF7
are converted to a mixture (M2) of compounds C1 and C5 via
synthetic pathway P1d wherein CF3 and CF7 are combined with
fragment F3 which attaches to the Y site on fragment F5 in CF3 and
CF7. The introduction of fragment F3 at site Y in CF3 and CF7
represents the third synthetic round (i.e. round n+2).
[0255] Yet another symbolic example of the one-pot generation of
mixtures of compounds, in accordance with the present invention, is
shown in FIG. 26. In silico generation of compounds commences with
the selection of fragment F7, which has three sites of attachment
(X, Y, and Z). This represents the first synthesis round (i.e.
round n). Next, F7 is subjected to synthetic pathway P1a wherein F7
is combined with fragment F2. F2 attaches to site X on fragment F7,
forming complex fragment CF1. At this stage, CF1 is subjected to
two synthetic pathways, P1b and P1b'. P1b employs fragment F1 which
is introduced onto site Y on CF1, thereby forming complex fragment
CF2, while P1b' employs fragment F3 which is introduced onto site Y
on CF1, thereby forming complex fragment CF8. Thus a mixture of
complex fragments (CF2 and CF8) are formed. Both fragments, F1 and
F3 can be introduced together (such as from a single reagent bottle
when actual synthesis is being undertaken) for the one-pot
generation of compounds if the chemistries associated with
introduction of these fragments into the compounds are compatible.
If not, these fragments can be introduced separately. Next, CF2 and
CF8 are subjected to synthetic pathway P1c wherein both complex
fragments are combined with fragment F5 which attaches to site Z on
CF2 and CF8, thereby forming complex fragments CF3 and CF9. The
formation of CF3 and CF9 completes the second synthesis round (i.e.
round n+1). As fragment F5 has two sites of attachment, site Y is
still available for attachment to another fragment. Therefore, CF3
is subjected to synthetic pathway P3 wherein CF3 is combined with
fragment F4. Introduction of F4 represents the third synthesis
round (i.e. round n+2). F4 is a mixture of fragments (and
introduced by adding a mixture of reagents), as shown in FIG. 22.
As a result, synthetic pathway P2 leads to the generation of
compounds C2 and C3. Simultaneously, CF9 combines with fragment F4,
via synthetic pathway P2', leading to the generation of compounds
C7 and C8. Thus mixture M3 is formed comprising compounds C2, C3,
C7 and C8.
[0256] The present invention also provides methods for the
generation of increasingly complex mixtures of compounds. An
example is shown in FIGS. 27a and 27b where mixture M4 is generated
and comprises sixteen compounds. The compounds in mixture M4 can be
generated by starting with fragments F7 and F8 in the first
synthesis round (i.e. round n). These fragments can then be
combined with fragment F2, which is introduced at site X in each
of.F7 and F8, forming complex fragment CF1 and CF5. Following this,
a mixture of fragments F1 and F3 are introduced into CF1 and CF5 at
site Y of these complex fragments, leading to the formation of four
complex fragments, CF2, CF6, CF8 and CF11. These complex fragments
are next combined with a mixture of fragments F5 and F6. Both F5
and F6 have two attachment sites such that site X on F5 and F6
attaches to site Z on CF2, CF6, CF8 and CF11 forming a mixture of
eight complex fragments, CF3, CF7, CF9, CF12, CF13, CF14, CF15 and
CF16. This completes the second synthesis round (i.e. round n+1).
As fragments F5 and F6 have two attachment sites, X and Y, the
abovementioned eight complex fragments have one more available
attachment site (i.e. site Y) onto which another fragment may be
introduced. Attachment of a fragment to site Y on these eight
complex fragments represents the third synthesis round (i.e. round
n+2). Next, fragment F4 is introduced into CF3, CF7, CF9, CF12,
CF13, CF14, CF15 and CF16. As fragment F4 is a mixture of two
constituent fragments, sixteen compounds are generated: C2, C3, C7,
C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19 and C20.
Thus it can be seen that by using multiple fragments in a one-pot
fashion and combining with mixtures of fragments, mixtures of
compounds of increasing complexity can be generated. The example in
FIGS. 27a and 27b shows sixteen unique compounds being generated as
mixture M4 when the library is generated by starting with two
fragments. It is recognized by the art-skilled that if the library
generation is commenced with more than two fragments or multiple
fragments are added to the same precursor fragment, even more
complex mixtures of compounds can be generated.
[0257] The present invention also provides methods for keeping
track of fragment addition in the various synthesis rounds. This
system of accounting is accomplished by tabulation of the synthesis
rounds which are correlated with addition of fragments. While for
the purposes of illustration of the invention, a tabulation method
of tracking fragment addition is described herein, it will be
recognized by the art-skilled that other algorithms, algorithmic
codes, computer readable mediums and various software coding
techniques know to those skilled in the computer arts may be used
for such tracking. The tables tracking fragment addition can be
used to produce structural representations of compounds and create
virtual libraries where actual synthesis of the compounds is not
desired. Tables tracking transformations, however, can be used to
synthesize compounds by selecting the appropriate transformations,
and in the case of multiple transformations, selecting the
preferable transformations to introduce the required fragment into
the compounds being synthesized.
[0258] FIG. 28 is descriptive of compound C1 in terms of the
fragments added in each synthesis round. The first synthesis round
(i.e. round n) commences with the selection of fragment F7. This is
followed by the sequential addition of fragments F2, F1 and F5 in
the second synthesis round (i.e. round n+1). Finally, compound C1
is generated by the addition of fragment F3 in the third synthesis
round (i.e. round n+2). The compounds thus generated can be stored
as a 2-dimensional virtual library, or may be converted to a
3-dimensional virtual library that can be used for in silico
docking to desired target molecules.
[0259] For the generation of virtual libraries of compounds and for
docking the library members onto target molecules, it suffices to
add compounds to the relational database in terms of its fragments
to track the addition of fragments in the various synthetic rounds.
However, when the actual synthesis of desired compounds of a
library is to be undertaken, it becomes necessary to specify the
actual synthetic steps, reagents, solvents, concentrations,
auxiliary compounds needed and other various synthetic factors in
order to effect such an actual synthesis of real chemical
compounds. Such synthetic steps, reagents, solvents, concentrations
and auxiliary compounds are, in fact, incorporated in to the above
described transformations. Thus by employing the concept of
transformations, the present invention provides methods to track
the compounds generated not only in terms of the fragments added
but as well as the synthetic parameters necessary for each
synthesis round.
[0260] FIG. 28 also shows the generation of compound C1 in terms of
the various transformations employed in the synthesis rounds. Four
synthesis pathways lead to the synthesis of compound C1 because of
the availability of multiple transformations that can introduce the
same fragment into the compound being synthesized. Thus, as seen in
FIG. 28, selection of fragment F7 constitutes transformation T9 in
the first synthesis round (i.e. round n). This is followed by the
addition of fragment F2 which is achieved by employing
transformation T2. Next, fragment F1 is added via transformation
T1. Fragment F5, however, may be added by employing either reagent
R6 via transformation T6 along synthesis paths 1 and 3, or reagent
R7 via transformation T7 along synthesis paths 2 and 4. Similarly,
the final fragment F3 can be added by using either reagent R3 via
transformation T3 along synthesis paths 1 and 2, or reagent R4 via
transformation T4 along synthesis paths 3 and 4. Thus FIG. 28 shows
that compound C1 can be actually synthesized via one of four
different synthetic schemes which can be tracked or tabulated and
accounted for using the methods of the present invention. Each of
the four tables is completely descriptive of each of the four
synthetic pathways for the preparation of C1. Thus, a user of the
present invention has available all the alternate pathways of
performing the same reaction (i.e. introducing the same fragment),
and can select the preferable or most appropriate synthetic route
to preparing the desired compounds.
[0261] FIG. 29 shows a similar transformation tracking table for
compounds C2 and C3 in mixture M1. Synthesis of compounds C2 and C3
commences with selection of fragment F7 which represents
transformation T9 (step 1 in FIG. 29) in the first synthesis round
(i.e. round n). Next, F7 is combined with fragment F2 via
transformation T2 in the second synthesis round (i.e. round n+1)
(step 2). In the same round, fragment F1, via transformation T1,
and fragment F5, via transformation T7 are added sequentially
(steps 3 and 4). Finally, fragment F4 is added in the third
synthesis round (i.e. round n+2). As F4 is a mixture of two
constituent fragments (because of two constituent reagents), the
table is duplicated at this stage (step 5) to account for the
different synthetic ways in which transformation T5 may be
accomplished (i.e. T5.sup.1 and T5.sup.2). Step 5 represents
compounds C2 and C3. Thus it can be seen that, in accordance with
the present invention, whenever there is more than one reagents
associated with a particular transformation, the table is
duplicated as many times as there are such reagents.
[0262] FIG. 30 shows a transformation tracking table for compounds
C1 and C5 in mixture M3. As the synthesis commences with two
fragments, F7 and F8, tracking begins with two parallel tables
(step 1 in FIG. 30). In the first synthesis round (i.e. round n),
F7 is selected via transformation T9, while F8 is selected via
transformation T10. The second synthesis round (i.e. round n+1)
commences at step 2 with the introduction of fragment F2 via
transformation T2. In step 3, transformation T1 introduces fragment
F1 into the compound. In step 4, transformation T7 introduces
fragment F5. This completes the second synthesis round (i.e. round
n+1). Finally, in the third synthesis round (i.e. round n+2),
transformation T4 is used to introduce fragment F3 (at step 5)
producing mixture M2 comprising compounds C1 and C5. In this
example, the tables are duplicated early in the synthetic scheme
because of the use of a mixture of fragments F7 and F8 at the
outset.
[0263] The transformation tracking table for compounds C2, C3, C7
and C8 of mixture M3 are shown in FIG. 31. The synthesis of these
compounds commences with the first synthesis round (i.e. round n)
in which fragment F7 is selected. This represents transformation T9
(shown in step 1 in FIG. 31). Step 2 in FIG. 31 depicts the second
synthesis round (i.e. round n+1) and involves the addition of
fragment F2 via transformation T2. While steps 1 and 2 involve
single transformations each, step 3 involves two different
transformations because two different fragments are being
introduced into the compounds through the use of two different
reagents. Therefore, at step 3 the table is twice duplicated
because two different reagents are being employed to introduce two
different fragments via two different transformations. In step 3,
transformation T1 is used to introduce fragment F1 while
transformation T3 is used to introduce fragment F3. The second
synthesis round (i.e. round n+1) is completed at step 4 with
transformation T7 which introduces fragment F5. In the final
synthesis round (i.e. the third round or round n+2), transformation
T5 is used to introduce fragment F4. As F4 is a mixture of two
constituent fragments, each table at step 5 is twice duplicated for
transformations T5.sup.1 and T5.sup.2 which represent each of the
constituent fragments of F4.
[0264] These figures represent merely one manner in which the
various fragments, reagents and transformations may be tracked
during the generation or synthesis of single compounds or mixtures
of compounds. It will, however, be recognized by the art-skilled
that various other algorithm schemes may be employed to track and
account for the fragments being introduced via transformations when
compounds are being generated in silico.
[0265] The libraries as described above as well as libraries
created by other means, can be synthesized on various automated
synthesizers. For illustrative purposes, the synthesizer utilized
for synthesis of above described libraries, is a variation of the
synthesizer described in U.S. Pat. Nos. 5,472,672 and 5,529,756,
the entire contents of which are herein incorporated by reference.
The synthesizer described in those patents was modified to include
movement in along the Y axis in addition to movement along the X
axis. As so modified, a 96-well array of compounds can be
synthesized by the synthesizer. The synthesizer can further include
temperature control and the ability to maintain an inert atmosphere
during all phases of a synthesis. The reagent array delivery format
employs orthogonal X-axis motion of a matrix of reaction vessels
and Y-axis motion of an array of reagents. Each reagent has its own
dedicated plumbing system to eliminate the possibility of
cross-contamination of reagents and line flushing and/or pipette
washing. This in combined with a high delivery speed obtained with
a reagent mapping system allows for the extremely rapid delivery of
reagents. This further allows long and complex reaction sequences
to be performed in an efficient and facile manner.
[0266] Software, as described below utilized in conduction with the
synthesizer allows the straightforward programming of the parallel
synthesis of a large number of compounds. The software utilizes a
general synthetic procedure in the form of a command (.cmd) file,
which calls upon certain reagents to be added to certain wells via
lookup in a sequence (.seq) file. The bottle position, flow rate,
and concentration of each reagent is stored in a lookup table
(.tab) file. Thus, once a synthetic method is outlined, a plate of
compounds is made by permutating a set of reagents, and writing the
resulting output to a text file. The text file is input directly
into the synthesizer and used for the synthesis of the plate of
compounds. The synthesizer can be interfaced with a relational
database allowing data output related to the synthesized compounds
to be registered in a highly efficient manner.
[0267] The seq, .cmd and tab files are built or constructed and
once constructed, are stored in an appropriate database. The .cmd
file is a synthesis file. This file can be built fresh to reflect a
completely new set of machine commands reflecting a set of chemical
synthesis steps (as for instance the above described
transformations) or it can modify an existing file stored in a
database by editing a stored file. The .cmd files are built using a
word processor and a command set of instructions as outlined
below.
[0268] In a like manner to the building the .cmd files, tab files
are built to reflect the necessary reagents used in the automatic
synthesizer for the particular chemistries necessary for the
library of desired compounds. Thus for each of a set of these
chemistries, a .tab file is built and stored in the database. As
with the .cmd files, an existing .tab file can be edited for use in
constructing a further .tab file.
[0269] Both the .cmd files and the tab files are linked together
for later retrieval from the database. Linking can be as simple as
using like file names to associate a .cmd file to its appropriate
tab file, e.g., syntheses.cmd is linked to syntheses.tab by use of
the same preamble in their names.
[0270] The automated, multi-well parallel array synthesizer employs
a reagent array delivery format, in which each reagent utilized has
a dedicated plumbing system. As seen in FIGS. 32 and 33, an inert
atmosphere 10 is maintained during all phases of a synthesis.
Temperature is controlled via a thermal transfer plate 12, which
holds an injection molded reaction block 14. The reaction plate
assembly slides in the X-axis direction, while eight nozzle blocks
(16, 18, 20, 22, 24, 26, 28 and 30) holding the reagent lines slide
in the Y-axis direction, allowing for the extremely rapid delivery
of any of 64 reagents to 96 wells. In addition, there are six banks
of fixed nozzle blocks (32, 34, 36, 38, 40 and 42) which deliver
the same reagent or solvent to eight wells at once, for a total of
72 possible reagents. In synthesizing compounds for screening, the
target reaction vessels, a 96 well plate 44 (a 2-dimensional
array), moves in one direction along the X axis, while the series
of independently controlled reagent delivery nozzles (16, 18, 20,
22, 24, 26, 28 and 30) move along the Y-axis relative to the
reaction vessel 46. As the reaction plate 44 and reagent nozzles
(16, 18, 20, 22, 24, 26, 28 and 30) can be moved independently at
the same time, this arrangement facilitated the extremely rapid
delivery of up to 72 reagents independently to each of the 96
reaction vessel wells.
[0271] The system software allows the straightforward programming
of the synthesis of a large number of compounds by supplying the
general synthetic procedure in the form of the command file to call
upon certain reagents to be added to specific wells via lookup in
the sequence file with the bottle position, flow rate, and
concentration of each reagent being stored in the separate reagent
table file. Compounds can be synthesized on various scales ranging
from small, as for example a 200 mmole scale, to larger scales, as
for example a 10 .mu.mole scale (3-5 mg). The resulting crude
compounds are generally >80% pure, and are utilized directly for
high throughput screening assays. Alternatively, prior to use the
plates can be subjected to quality control to ascertain their exact
purity. Use of the synthesizer results in a very efficient means
for the parallel synthesis of compounds for screening.
[0272] The software inputs accept tab delimited text files from any
text editor. A typical command file, a .cmd file, is shown in
Example 5, Table 3. A typical sequence file, a seq files, is shown
in Example 5, Table 4, and a typical reagent file, a .tab file, is
shown in Example 5, Table 5. Typically some of the wells of the 96
well plate may be left empty (depending on the number of compounds
in the individual synthesis) or some of the well may have compounds
that will serve as standards for comparison or analytical
purposes.
[0273] Prior to loading reagents, moisture sensitive reagent lines
are purged with argon at 10 for 20 minutes. Reagents are dissolved
to appropriate concentrations and installed on the synthesizer.
Large bottles, collectively identified as 46 in FIG. 32 (containing
8 delivery lines) are used for wash solvents and the delivery of
general activators, cleaving reagents and other reagents that may
be used in multiple wells during any particular synthesis. Small
septa bottles, collectively identified as 48 in FIG. 32, are
utilized to contain individual reagent compounds. This allows for
anhydrous preparation and efficient installation of multiple
reagents by using needles to pressurize the bottle, and as a
delivery path. After all reagents are installed, the lines are
primed with reagent, flow rates measured, then entered into the
reagent table (.tab file). A dry resin loaded plate is removed from
vacuum and installed in the machine for the synthesis.
[0274] The modified 96 well polypropylene plate 44 is utilized as
the reaction vessel. The working volume in each well is
approximately 700 .mu.l. The bottom of each well is provided with a
pressed-fit 20 .mu.m polypropylene frit and a long capillary exit
into a lower collection chamber as is illustrated in FIG. 5 of the
above referenced U.S. Pat. No. 5,372,672. The solid support for use
in holding the growing compounds during synthesis is loaded into
the wells of the synthesis plate 44 by pipetting the desired volume
of a balanced density slurry of the support suspended in an
appropriate solvent, typically an acetonitrile-methylene chloride
mixture. Reactions can be run on various scales as for instance the
above noted 200 mmole and 10 .mu.mol scales. Various supports can
be utilized for synthesis. Particularly useful supports include
medium loading polystyrene-PEG supports such as TentaGel.TM. or
ArgoGel.TM..
[0275] As seen in FIG. 33, the synthesis plate is transported back
and forth in the X-direction under an array of 8 moveable banks
(16, 18, 20, 22, 24, 26, 28 and 30) of 8 nozzles (64 total) in the
Y-direction, and 6 banks (32, 34, 36, 38, 40 and 42) of 48 fixed
nozzles, so that each well can receive the appropriate amounts of
reagents and/or solvents from any reservoir (large bottle or
smaller septa bottle). A sliding balloon-type seal 50 surrounds
this nozzle array and joins it to the reaction plate headspace 52.
A slow sweep of nitrogen or argon 20 at ambient pressure across the
plate headspace is used to preserve an anhydrous environment.
[0276] The liquid contents in each well do not drip out until the
headspace pressure exceeds the capillary forces on the liquid in
the exit nozzle. A slight positive pressure in the lower collection
chamber can be added to eliminate residual slow leakage from filled
wells, or to effect agitation by bubbling inert gas through the
suspension. In order to empty the wells, the headspace gas outlet
valve is closed and the internal pressure raised to about 2 psi.
Normally, liquid contents are blown directly to waste 54. However,
a 96 well microtiter plate can be inserted into the lower chamber
beneath the synthesis plate in order to collect the individual well
eluent for spectrophotometric monitoring of reaction progress and
yield.
[0277] The basic plumbing scheme for the machine is the
gas-pressurized delivery of reagents. Each reagent is delivered to
the synthesis plate through a dedicated supply line, collectively
identified at 56, solenoid valve collectively identified at 58 and
nozzle, collectively identified at 60. Reagents never cross paths
until they reach the reaction well. Thus, no line needs to be
washed or flushed prior to its next use and there is no possibility
of cross-contamination of reagents. The liquid delivery velocity is
sufficiently energetic to thoroughly mix the contents within a well
to form a homogeneous solution, even when employing solutions
having drastically different densities. With this mixing, once
reactants are in homogeneous solution, diffusion carries the
individual components into and out of the solid support matrix
where the desired reaction takes place. Each reagent reservoir can
be plumbed to either a single nozzle or any combination of up to 8
nozzles. Each nozzle is also provided with a concentric nozzle
washer to wash the outside of the delivery nozzles in order to
eliminate problems of crystallized reactant buildup due to slow
evaporation of solvent at the tips of the nozzles. The nozzles and
supply lines can be primed into a set of dummy wells directly to
waste at any time.
[0278] The entire plumbing system is fabricated with Teflon tubing,
and reagent reservoirs are accessed via syringe needle/septa or
direct connection into the higher capacity bottles. The septum
vials 48 are held in removable 8-bottle racks to facilitate easy
setup and cleaning. The priming volume for each line is about 350
.mu.l. The minimum delivery volume is about 2 .mu.l, and flow rate
accuracy is .+-.5%. The actual amount of material delivered depends
on a timed flow of liquid. The flow rate for a particular solvent
will depend on its viscosity and wetting characteristics of the
Teflon tubing. The flow rate (typically 200-350 .mu.l per sec) is
experimentally determined, and this information is contained in the
reagent table setup file.
[0279] Heating and cooling of the reaction block 14 is effected
utilizing a recirculating heat exchanger plate 12, similar to that
found in PCR thermocyclers, that nests with the polypropylene
synthesis plate 44 to provide good thermal contact. The liquid
contents in a well can be heated or cooled at about 10.degree. C.
per minute over a range of +5 to +80.degree. C., as polypropylene
begins to soften and deform at about 80.degree. C. For temperatures
greater than this, a non-disposable synthesis plate machined from
stainless steel or monel with replaceable frits might be
utilized.
[0280] The hardware controller is designed around a set of three 1
MHZ 86332 chips. This controller is used to drive the single X-axis
and 8 Y-axis stepper motors as well as provide the timing functions
for a total of 154 solenoid valves. Each chip has 16 bidirectional
timer I/O and 8 interrupt channels in its timer processing unit
(TPU). These are used to provide the step and direction signals,
and to read 3 encoder inputs and 2 limit switches for controlling
up to three motors per chip. Each 86332 chip also drives a serial
chain of 8 UNC5891A darlington array chips to provide power to 64
valves with msec resolution. The controller communicates with the
Windows software interface program running on a PC via a 19200 Hz
serial channel, and uses an elementary instruction set to
communicate valve_number and time_open, and motor_number and
position_data.
[0281] The three components of the software program that run the
array synthesizer, the generalized procedure or command (.cmd) file
which specifies the synthesis instructions to be performed, the
sequence (.seq) file which specifies the scale of the reaction and
the order in which variable groups will be added to the core
synthon, and the reagent table (.tab) file which specifies the name
of a chemical, its location (bottle number), flow rate, and
concentration are utilized in conjunction with a basic set of
command instructions.
[0282] The basic set of command instructions are:
2 ADD IF {block of instructions} END_IF REPEAT {block of
instructions} END_REPEAT PRIME, NOZZLE_WASH WAIT, DRAIN LOAD,
REMOVE NEXT_SEQUENCE LOOP_BEGIN, LOOP_END
[0283] The ADD instruction has two forms, and is intended to have
the look and feel of a standard chemical equation. Reagents are
specified to be added by a molar amount if the number proceeds the
name identifier, or by an absolute volume in micro liters if the
number follows the identifier. The number of reagents to be added
is a parsed list, separated by the `+` sign. For variable reagent
identifiers, the key word, <seq>, means look in the sequence
table for the identity of the reagent to be added, while the key
word, <act>, means add the reagent which is associated with
that particular <seq>. Reagents are delivered in the order
specified in the list.
[0284] Thus:
[0285] ADD ACN 300
[0286] means: Add 300 .mu.l of the named reagent ACN to each well
of active synthesis
[0287] ADD <seq>300
[0288] means: If the sequence pointer in the seq file is to a
reagent in the list of reagents, independent of scale, add 300
.mu.l of that particular reagent specified for that well.
[0289] ADD 1.1 PYR+1.0<seq>+1.1<act1>
[0290] means: If the sequence pointer in the seq file is to a
reagent in the list of acids in the Class ACIDS.sub.--1, and PYR is
the name of pyridine, and ethyl chloroformate is defined in the tab
file to activate the class, ACIDS.sub.--1, then this instruction
means:
[0291] Add
[0292] 1.1 equiv. pyridine
[0293] 1.0 equiv. of the acid specified for that well and
[0294] 1.1 equiv. of the activator, ethyl chloroformate
[0295] The IF command allows one to test what type of reagent is
specified in the <seq>variable and process the succeeding
block of commands accordingly.
[0296] Thus:
3 ACYLATION {the procedure name} BEGIN IF CLASS = ACIDS_1 ADD 1.0
<seq> + 1.1 <act1> + 1.1 PYR WAIT 60 ENDIF IF CLASS =
ACIDS_2 ADD 1.0 <seq> + 1.2 <act1> + 1.2 TEA ENDIF WAIT
60 DRAIN 10 END
[0297] means: Operate on those wells for which reagents contained
in the Acid.sub.--1 class are specified, WAIT 60 sec, then operate
on those wells for which reagents contained in the Acid.sub.--2
class are specified, then WAIT 60 sec longer, then DRAIN the whole
plate. Note that the Acid.sub.--1 group has reacted for a total of
120 sec, while the Acid.sub.--2 group has reacted for only 60
sec.
[0298] The REPEAT command is a simple way to execute the same block
of commands multiple times.
[0299] Thus:
4 WASH_1 {the procedure name} BEGIN REPEAT 3 ADD ACN 300 DRAIN 15
END_REPEAT END
[0300] means: repeats the add acetonitrile and drain sequence for
each well three times.
[0301] The PRIME command will operate either on specific named
reagents or on nozzles which will be used in the next associated
<seq>operation. The .mu.l amount dispensed into a prime port
is a constant that can be specified in a config.dat file.
[0302] The NOZZLE_WASH command for washing the outside of reaction
nozzles free from residue due to evaporation of reagent solvent
will operate either on specific named reagents or on nozzles which
have been used in the preceding associated <seq>operation.
The machine is plumbed such that if any nozzle in a block has been
used, all the nozzles in that block will be washed into the prime
port.
[0303] The WAIT and DRAIN commands are by seconds, with the drain
command applying a gas pressure over the top surface of the plate
in order to drain the wells.
[0304] The LOAD and REMOVE commands are instructions for the
machine to pause for operator action.
[0305] The NEXT_SEQUENCE command increments the sequence pointer to
the next group of substituents to be added in the sequence
file.
[0306] The general form of a .seq file entry is the definition:
[0307] Well_No Well_ID Scale Sequence
[0308] The sequence information is conveyed by a series of columns,
each of which represents a variable reagent to be added at a
particular position. The scale (.mu.mole) variable is included so
that reactions of different scale can be run at the same time if
desired. The reagents are defined in a lookup table (the .tab
file), which specifies the name of the reagent as referred to in
the sequence and command files, its location (bottle number), flow
rate, and concentration. This information is then used by the
controller software and hardware to determine both the appropriate
slider motion to position the plate and slider arms for delivery of
a specific reagent, as well as the specific valve and time required
to deliver the appropriate reagents. The adept classification of
reagents allows the use of conditional IF loops from within a
command file to perform addition of different reagents differently
during a `single step` performed across 96 wells simultaneously.
Reagents can be group according to "class." Thus all for a
particular synthesis that utilizes a fragment that is based on
amino acids, the class "AMINO_ACIDS" can be created. The special
class ACTIVATORS defines certain reagents that always get added
with a particular class of reagents (for example Betaine utilized
to activate the class AMINO_ACIDS).
[0309] The general form of the .tab file is the definition:
[0310] Class Bottle Reagent Name Flow_rate Conc.
[0311] The LOOP_BEGIN and LOOP_END commands define the block of
commands which will continue to operate until a NEXT_SEQUENCE
command points past the end of the longest list of reactants in any
well.
[0312] Not included in the command set is a MOVE command. For all
of the above commands, if any plate or nozzle movement is required,
this is automatically executed in order to perform the desired
solvent or reagent delivery operation. This is accomplished by the
controller software and hardware, which determines the correct
nozzle(s) and well(s) required for a particular reagent addition,
then synchronizes the position of the requisite nozzle and well
prior to adding the reagent.
[0313] A MANUAL mode is also utilized in which the synthesis plate
and nozzle blocks can be `homed` or moved to any position by the
operator, the nozzles primed or washed, the various reagent bottles
depressurized or washed with solvent, the chamber pressurized, etc.
The automatic COMMAND mode can be interrupted at any point, MANUAL
commands executed, and then operation resumed at the appropriate
location. The sequence pointer can be incremented to restart a
synthesis anywhere within a command file.
[0314] The compounds to be synthesized can be rearranged or grouped
for optimization of synthesis. Such grouping can be effected based
on any parameter that will result in optimization of synthesis. One
such factor considers the fragment of the compounds that are
directly linked to the supporting resin. If the same fragment is to
be utilized multiple times, it can be joined to the support in a
batch wise manner and aliquots of this batch synthesis then loaded
into the individual wells of the plate prior to start of the
synthesis. Another parameter is by positioning like compounds near
each other. By grouping like fragments near each other, machine
movements are conserved and in doing so, overall synthesis time is
shortened.
[0315] In utilizing the multi well format for compound synthesis,
for each compound to be synthesized, an aliquot of a solid support
bearing the proper first fragment thereon can be added to the well
for synthesis. Thus prior to loading the sequence of compounds to
be synthesized in the .seq file, they are sorted by this fragment.
Based on that sorting, all of compounds having similar first
fragments are positioned together in adjacent wells on the plate.
Thus in loading the fragment-bearing solid support into the
synthesis wells, machine movements are conserved. In a further
method of preparing compounds, only the solid support is added to
the wells and the first fragment is then linked to the solid
support as the first synthetic step. The seq file is appropriately
modified to reflect that the first segment is to be added.
[0316] Once sorted into types, the position of the compounds on the
synthesis plates is specified by the creation of a .seq file as
described above. The seq file is associated with the respective
.cmd and tab files needed for synthesis of the particular
chemistries specified for the compounds by retrieval of the .cmd
and .tab files a database. These files are then input into the
multi well synthesizer for compound synthesis. Upon completion of
synthesis, for shipping, storage or other handling purposes, the
plates can be lyophilized at this point if desired. Upon
lyophilization, each well contains the compounds located therein as
a dry compound.
[0317] To illustrate the invention, a synthetic was effected
utilizing the methods of the invention to generate a small library
(.about.1200) of discreet hydroxamic acids. The total library is
shown in Table 2 below. Two distinct chemical pathways were
utilized for the automated synthesis of the illustrative library of
hydroxamic acid compounds. These are shown in FIGS. 34 and 35. Each
pathway had its own advantages.
[0318] The illustrative hydroxamic library compounds generally
correspond in structure to compound CI of FIG. 14, formed from a
hydroxylamine fragment, a valine fragment (the amino acid fragment)
and a sulfonyl-4-methoxybenzene fragment (the sulfonyl fragment) of
FIG. 15. They differ from one another with respect to their amino
acid fragment and their sulfonyl fragment. They have in common
their hydroxyl amine fragment. Compound CI directly corresponds
(they are one in the same) to compound a-x of Table 2. These
compounds further corresponds to symbolic compound CI'.
[0319] For illustrative purposes to demonstrate complex chemical
structures and mixtures, the symbolic tables shown in the FIGS. 28,
29, 30, and 31 describe certain complex symbolic structures and
thus equally complex chemical structures. Compared to these complex
structures and mixtures, compound CI' is less complex, however, its
construction embodies the same principles as used to describe the
structures of those figures. Since it embodies the same principles,
one can construct a similar table for compound CI'. Thus in round n
it would have the fragment Fi', in round n+1 the fragment Fii' and
in round n+2, the fragment Fiii'. A transformation table can
likewise be constructed listing Ti in round n, Tii in round n+1 and
Tiii in round n+2. This information is then used to instruct the
automated synthesizer to prepare the actual library.
[0320] In constructing the illustrative hydroxamic library
utilizing the synthetic pathway of FIG. 35, the first fragment, the
hydroxyl amine fragment is the same in all members of the library.
Therefore, for ease of synthesis, it is added already attached to a
solid support to wells in a synthesis plate. This reduces the
complexity of the synthesis by a factor of "one fragment" and in
turn reduce the number of rounds by one of synthesis that must be
effected on the synthesizer. In essence this eliminates the round n
as described in the tables of FIGS. 28, 29, 30 and 31.
[0321] As described above, the general form of a seq file entry
was:
[0322] Well_No Well_ID Scale Sequence
[0323] where the "Sequence" information was conveyed by a series of
columns. Since the round n transformation has been generalized for
each well on the plate by adding the hydroxyl amine fragment
attached to a solid support, only two Sequence columns are
necessary to describe the synthesis, one for the round n+1 showing
the amino acid reagent used and one for the round n+2 showing the
sulfonyl reagent used. Each "Sequence" column corresponds to a
reagent which is a member of a transformation represented in the
tracking tables. This reagent is linked by the one to one
relationship specified by the transformation to its resulting
fragment.
[0324] Various algorithms, as will be evident to those skilled in
the computer programing arts, could be utilized to convert the
information contained withing the tracking tables described above
into the format suitable for synthesis utilizing the parallel array
synthesizer described herein. One preferred way to accomplish this
is by looking up the transformation required for each particular
round of synthesis for each compound or group of compounds in the
tracking table. The appropriate complex or single reagent is then
written to a software file in a format such that each reagent
utilized for the transformation indicated in the tracking table at
the appropriate synthesis round corresponds to a single column
entry in the seq file. The compounds or groups of compounds stored
in the database and their location in the reaction vessel in which
they are synthesized are then linked by the Well_ID field of the
seq file, which is assigned by the database. Thus, having described
the compounds by their transformations allows for facile
construction of the seq file need for synthesis. This is
illustrated by the synthesis files for the parallel array
synthesizer detailed above, but the process is equally applicable
to any suitable programmable chemical synthesis apparatus.
[0325] In a like manner the general form of the tab file was:
5 Class Bottle Reagent Flow Conc. Name Rate
[0326] Here complex or single reagents can be specified in the
"Reagent Name" as defined by the bottle the reagent or mixture of
reagents is located. Whether it was a single reagent or a complex
reagent mixture specified by a particular transformation, that
information is carried over to the synthesizer instructions by the
appropriate entry in the .tab file for that reagent. As for the seq
file creation, the information in the transformation tracking table
can be readily converted to a tab file. Each complex or single
reagent called for in the synthesis is given a line entry in the
.tab file. Additionally, the single reagent components of complex
reagents may be specified in a comments section of the .tab file to
facilitate preparation of complex reagents. The appropriate
conditions for the specified reagent as indicated in the
corresponding transformation are also written to the proscribed
field in the .tab file. Additionally, associated reagents for
accomplishing the specified transformation (such as activators,
bases, scavangers, coupling reagents, etc.) may also be written to
the .tab file as appropriate. In the synthesis of the illustrative
hydroxamic acid library, the activator named "betaine" is
associated with the transformation attaching the amino acid to
solid support. It is placed in the tab file, along with a modifier
specifing which reagents it is associated. As a result of having
described the compounds by their transformations, construction of
the .tab file need for synthesis is facilitate. This is illustrated
by the synthesis files for the parallel array synthesizer detailed
herein, but the process is equally applicable to any suitable
programmable chemical synthesis apparatus.
[0327] As the complexity of the fragments for the compounds in a
library increases as for instances steps P1a, P1b, P1c, P1c and P3b
of FIG. 24, they in turn require more column entries the "Sequence"
portion of seq. However, if complexity is achieved by using
mixtures of reagents that are used in unison, as for instances step
P2 of FIG. 24, this is controlled by locating them in a single
reagent bottle as specified by the .tab file.
[0328] In reference again to the illustrative hydoxamic acid
library of Table 2, the first method of synthesis, illustrated in
FIG. 34, entails derivatizing commercially available ArgoGel-OH.TM.
(which has an PEG based alcohol as the reactive functional group)
with an FMOC-amino acid via a modified Mitsunobu reaction employing
the sulfonamide betaine 1 as the activating species. This reaction
proceeded to essentially 100% completion (by FMOC) in several
hours, and has the advantage over other loading procedure
(symmetric anhydride/DMAP) of eliminating the potential for
racemization of the amino acid. It also requires less equivalents,
as one equivalent of amino acid is not wasted due to the formation
of a symmetric anhydride, and the potential for FMOC loss is
minimized. The resin bound ester 2 was next deprotected, then
sulfonylated using a sulfonyl chloride in pyridine. The yield of
the Mitsunobu loading step was measured by collecting the washes
from the FMOC deprotection, followed by spectrophotometric
determination of the amount released in a 96 well plate reader.
This information was then written to a data file for import into a
database, which allows a yield estimate of the synthesized
compounds. It was found that cleavage of the ester 4 with
hydroxylamine in 1,4-dioxane (50% aqueous NH.sub.2OH diluted to 4 M
final NH.sub.2OH concentration with 1,4-dioxane) generally
proceeded to completion overnight at room temperature to provide
the desired hydroxamic acids 5. A small amount (10-20%) of the
corresponding carboxylic acid resulted from competitive hydrolysis
for hindered amino acids such as valine, even when anhydrous
hydroxylamine was employed. Several hindered amino acids and
electron deficient sulfonyl chlorides failed completely with this
method as indicated in Table 2 below.
[0329] The procedure has the advantage that orthogonal deprotection
and cleavage strategies can be employed, allowing standard peptide
acid labile side chain protection (t-butyl based, trityl, PMC,
etc.) to be used on the amino acid component. This allows isolation
of products free from side chain protection by-products in the case
of commonly used trityl and sulfonyl based protection of histidine,
arginine, glutamine, and asparagine. Thus, the resin bound ester 4
can be treated with anhydrous TFA for 4 h on the instrument,
resulting in complete side chain deprotection. If cleaned of TFA
immediately after synthesis, the instrument, including lines and
valves were unaffected by the extreme conditions. The support could
then be washed and the product 5 cleaved from support using the
standard methodology. This synthesis was accomplished very readily
on the automated parallel array synthesizer, using a very simple
command file, which functions as a `general procedure`.
Representative command, sequence and tab files are detailed in the
Example 5 below to illustrate the synthesis.
[0330] The second method utilized the acid labile Wang based
hydroxylamine support 6 (FIG. 35) to circumvent the minor problem
of competitive hydrolysis, and the failure of electron deficient
sulfonyl chlorides. The resin was prepared in an analogous manner
to the procedure described by Atheron et al., Solid Peptide
Synthesis: A Practical Approach; IRL Press: Oxford, UK 1989: p 135
employing an initial Mitsunobu reaction of ArgoGel-Wang.TM. resin
with N-hydroxyphthalimide, followed by deprotection with
methylhydrazine to afford 6 in quantitative yield by gel-phase
.sup.13C NMR. The hydroxylamine resin was then acylated with an
FMOC-amino acid utilizing standard peptide coupling methodology to
provide 7, which was deprotected then sulfonylated as before to
provide resin bound hydroxamic acid 8. This material was
efficiently cleaved from the resin with TFA containing Et.sub.3SiH
(5% v/v) as a scavenger to provide compounds 5.
[0331] A molecular interaction site is a region of a nucleic acid
which has secondary structure. Preferably, the molecular
interaction site is conserved between a plurality of different
taxonomic species. The nucleic acid can be either eukaryotic or
prokaryotic. The nucleic acid is preferably mRNA, pre-mRNA, tRNA,
rRNA, or snRNA. The RNA can be viral, fungal, parasitic, bacterial,
or yeast. Preferably, the molecular interaction site is present in
a region of an RNA which is highly conserved among a plurality of
taxonomic species. In accordance with some preferred embodiments of
this invention, it will be appreciated that the biomolecules having
a molecular interaction site or sites, especially RNAs, may be
derived from a number of sources. Thus, such RNA targets can be
identifed by any means, rendered into three dimensional
representations and employed for the identification of compounds
which can interact with them to effect modulation of the RNA.
[0332] The three dimensional structure of a molecular interaction
site, preferably of an RNA, can be manipulated as a numerical
representation. Computer software that provides one skilled in the
art with the ability to design molecules based on the chemistry
being performed and on available reaction building blocks is
commercially available. Software packages from companies such as,
for example, Tripos (St. Louis, Mo.), Molecular Simulations (San
Diego, Calif.), MDL Information Systems (San Leandro, Calif.) and
Chemical Design (NJ) provide means for computational generation of
structures. These software products also provide means for
evaluating and comparing computationally generated molecules and
their structures. In silico collections of molecular interaction
sites can be generated using the software from any of the
above-mentioned vendors and others which are or may become
available.
[0333] A set of structural constraints for the molecular
interaction site of the RNA can be generated from biochemical
analyses such as, for example, enzymatic mapping and chemical
probes, and from genomics information such as, for example,
covariance and sequence conservation. Information such as this can
be used to pair bases in the stem or other region of a particular
secondary structure. Additional structural hypotheses can be
generated for noncanonical base pairing schemes in loop and bulge
regions. A Monte Carlo search procedure can sample the possible
conformations of the RNA consistent with the program constraints
and produce three dimensional structures.
[0334] Reports of the generation of three dimensional, in silico
representations are available from the standpoint of library
design, generation, and screening against protein targets.
Likewise, some efforts in the area of generating RNA models have
been reported in the literature. However, there are no reports on
the use of structure-based design approaches to query in silico
representations of organic molecules, "small" molecules,
oligonucleotides or other nucleic acids, with three dimensional, in
silico, representations of RNA structures. The present invention
preferably employs computer software that allows the construction
of three dimensional models of RNA structure, the construction of
three dimensional, in silico representations of a plurality of
organic compounds, "small" molecules, polymeric compounds,
oligonucleotides and other nucleic acids, screening of such in
silico representations against RNA molecular interaction sites in
silico, scoring and identifying the best potential binders from the
plurality of compounds, and finally, synthesizing such compounds in
a combinatorial fashion and testing them experimentally to identify
new ligands for such targets.
[0335] In preferred embodiments of the invention, an automated
computational search algorithm, such as those described above, is
used to predict all of the allowed three dimensional molecular
interaction site structures, preferably from RNA, which are
consistent with the biochemical and genomic constraints specified
by the user. Based e.g. on their root-mean-squared deviation
values, these structures are clustered into different families. A
representative member or members of each family can be subjected to
further structural refinement via molecular dynamics with explicit
solvent and cations.
[0336] Structural enumeration and representation by these software
programs is typically done by drawing molecular scaffolds and
substituents in two dimensions. Once drawn and stored in the
computer, these molecules may be rendered into three dimensional
structures using algorithms present within the commercially
available software. Preferably, MC-SYM is used to create three
dimensional representations of the molecular interaction site. The
rendering of two dimensional structures of molecular interaction
sites into three dimensional models typically generates a low
energy conformation or a collection of low energy conformers of
each molecule. The end result of these commercially available
programs is the conversion of a nucleic acid sequence containing a
molecular interaction site into families of similar numerical
representations of the three dimensional structures of the
molecular interaction site. These numerical representations form an
ensemble data set.
[0337] The three dimensional structures of a plurality of
compounds, preferably "small" organic compounds, can be designated
as a compound data set comprising numerical representations of the
three dimensional structures of the compounds.
[0338] "Small" molecules in this context refers to non-oligomeric
organic compounds. Two dimensional structures of compounds can be
converted to three dimensional structures, as described above for
the molecular interaction sites, and used for querying against
three dimensional structures of the molecular interaction sites.
The two dimensional structures of compounds can be generated
rapidly using structure rendering algorithms commercially
available. The three dimensional representation of the compounds
which are polymeric in nature, such as oligonucleotides or other
nucleic acids structures, may be generated using the literature
methods described above. A three dimensional structure of "small"
molecules or other compounds can be generated and a low energy
conformation can be obtained from a short molecular dynamics
minimization. These three dimensional structures can be stored in a
relational database. The compounds upon which three dimensional
structures are constructed can be proprietary, commercially
available, or virtual.
[0339] In some preferred embodiments of the invention, a compound
data set comprising numerical representations of the three
dimensional structure of a plurality of organic compounds is
provided by, for example, Converter (MSI, San Diego) from two
dimensional compound libraries generated by, for example, a
computer program modified from a commercial program. Other suitable
databases can be constructed by converting two dimensional
structures of chemical compounds into three dimensional structures,
as described above. The software is described in greater detail
elsewhere in this U.S. Application. The end result is the
conversion of a two dimensional structure of organic compounds into
numerical representations of the three dimensional structures of a
plurality of organic compounds. These numerical representations are
presented as a compound data set.
[0340] After both the numerical representations of the
three-dimensional structure of the molecular interaction sites and
the compound data set comprising numerical representations of the
three dimensional structures of a plurality of organic compounds
are obtained, the numerical representations of the molecular
interaction sites are compared with members of the compound data
set to generate a hierarchy of the organic compounds. The hierarchy
is ranked in accordance with the ability of the organic compounds
to form physical interactions with the molecular interaction site.
Preferably, the comparing is carried out seriatim upon the members
of the compound data set. In accordance with some embodiments, the
comparison can be performed with a plurality of molecular
interaction sites at the same time.
[0341] A variety of theoretical and computational methods are known
by those skilled in the art to study and optimize the interactions
of "small" molecules or organic compounds with biological targets
such as nucleic acids. These structure-based drug design tools have
been very useful in modeling the interactions of proteins with
small molecule ligands and in optimizing these interactions.
Typically this type of study has been performed when the structure
of the protein receptor was known by querying individual small
molecules, one at a time, against this receptor. Usually these
small molecules had either been co-crystallized with the receptor,
were related to other molecules that had been co-crystallized or
were molecules for which some body of knowledge existed concerning
their interactions with the receptor. A significant advance in this
area was the development of a software program called DOCK that
allows structure-based database searches to find and identify
molecules that are expected to bind to a receptor of interest.
Kuntz, et al., Acc. Chem. Res., 1994, 27, 117, and Gschwend and
Kuntz, J. Compt.-Aided Mol. Des., 1996, 10, 123. DOCK 4.0 is
commercially available from the Regents of the University of
California. Equivalent programs are also comprehended in the
present invention. DOCK allows the screening of a large collection
of molecules whose three dimensional structures have been generated
in silico, i.e., in computer readable format, but for which no
prior knowledge of interactions with the ligands is available.
DOCK, therefore, is a significant tool to the process of
discovering new ligands to a molecule of interest and is presently
preferred for use herein.
[0342] The DOCK program has been widely applied to protein targets
and the identification of ligands that bind to them. Typically, new
classes of molecules that bind to known targets have been
identified, and later verified by in vitro experiments. The DOCK
software program consists of several modules, including SPHGEN
(Kuntz, et al., J. Mol. Biol., 1982, 161, 269) and CHEMGRID (Meng,
et al., J. Conput. Chem., 1992, 13, 505). SPHGEN generates clusters
of overlapping spheres that describe the solvent-accessible surface
of the binding pocket within the target receptor. Each cluster
represents a possible binding site for small molecules. CHEMGRID
precalculates and stores in a grid file the information necessary
for force field scoring of the interactions between binding
molecule and target. The scoring function approximates molecular
mechanics interaction energies and consists of van der Waals and
electrostatic components. DOCK uses the selected cluster of spheres
to orient ligands molecules in the targeted site on the receptor.
Each molecule within a previously generated three dimensional
database is tested in thousands of orientations within the site,
and each orientation is evaluated by the scoring function. Only
that orientation with the best score for each compound so screened
is stored in the output file. Finally, all compounds of the
database are ranked in a hierarchy in order of their scores and a
collection of the best candidates may then be screened
experimentally.
[0343] Using DOCK, numerous ligands have been identified for a
variety of protein targets. Recent efforts in this area have
resulted in reports of the use-of DOCK to identify and design small
molecule ligands that exhibit binding specificity for nucleic acids
such as RNA double helices. While RNA plays a significant role in
many diseases such as AIDS, viral and bacterial infections, few
studies have been made on small molecules capable of specific RNA
binding. Compounds possessing specificity for the RNA double helix,
based on the unique geometry of its deep major groove, were
identified using the DOCK methodology. Chen, et al., Biochemistry,
1997, 36, 11402 and Kuntz, et al., Acc. Chem. Res., 1994, 27, 117.
Recently, the application of DOCK to the problem of ligand
recognition in DNA quadruplexes has been reported. Chen, et al.,
Proc. Natl. Acad. Sci., 1996, 93, 2635.
[0344] Preferably, individual compounds are designated as mol
files, for example, and combined into a collection of in silico
representations using an appropriate chemical structure program or
equivalent software. These two dimensional mol files are exported
and converted into three dimensional structures using commercial
software such as Converter (Molecular Simulations Inc., San Diego)
or equivalent software, as described above. Atom types suitable for
use with a docking program such as DOCK are assigned to all atoms
in the three dimensional mol file using software such as, for
example, Babel, or with other equivalent software.
[0345] A low-energy conformation of each molecule is generated with
software such as Discover (MSI, San Diego). An orientation search
is performed by bringing each compound of the plurality of
compounds into proximity with the molecular interaction site in
many orientations using DOCK. A contact score is determined for
each orientation, and the optimum orientation of the compound is
subsequently used. Alternatively, the conformation of the compound
can be determined from a template conformation of the scaffold
determined previously.
[0346] The interaction of a plurality of compounds and molecular
interaction sites is examined by comparing the numerical
representations of the molecular interaction sites with members of
the compound data set. Preferably, a plurality of compounds such as
those generated by a computer program or otherwise, is compared to
the molecular interaction site and undergoes random "motions" among
the dihedral bonds of the compounds. Preferably about 20,000 to
100,000 compounds are compared to at least one molecular
interaction site. Typically, 20,000 compounds are compared to about
five molecular interaction sites and scored. Individual
conformations of the three dimensional structures are placed at the
target site in many orientations. Moreover, during execution of the
DOCK program, the compounds and molecular interaction sites are
allowed to be "flexible" such that the optimum hydrogen bonding,
electrostatic, and van der Waals contacts can be realized. The
energy of the interaction is calculated and stored for 10-15
possible orientations of the compounds and molecular interaction
sites.
[0347] The relative weights of each energy contribution are updated
constantly to insure that the calculated binding scores for all
compounds reflect the experimental binding data. The binding energy
for each orientation is scored on the basis of hydrogen bonding,
van der Waals contacts, electrostatics, solvation/desolvation, and
the quality of the fit. The lowest-energy van der Waals, dipolar,
and hydrogen bonding interactions between the compound and the
molecular interaction site are determined, and summed. In preferred
embodiments, these parameters can be adjusted according to the
results obtained empirically. The binding energies for each
molecule against the target are output to a relational database.
The relational database contains a hierarchy of the compounds
ranked in accordance with the ability of the compounds to form
physical interactions with the molecular interaction site. The
higher ranked compounds are better able to form physical
interactions with the molecular interaction site.
[0348] In a preferred embodiment, the highest ranking, i.e., the
best fitting compounds, are selected for synthesis. In preferred
embodiments of the invention, those compounds which are likely to
have desired binding characteristics based on binding data are
selected for synthesis. Preferably the highest ranking 5% are
selected for synthesis. More preferably, the highest ranking 10%
are selected for syntheses. Even more preferably, the highest
ranking 20% are selected for synthesis. The synthesis of the
selected compounds can be automated using a parallel array
synthesizer or prepared using solution-phase or other solid-phase
methods and instruments. In addition, the interaction of the highly
ranked compounds with the nucleic acid containing the molecular
interaction site is assessed as described below.
[0349] The interaction of the highly ranked organic compounds with
the nucleic acid containing the molecular interaction site can be
assessed by numerous methods known to those skilled in the art. For
example, the highest ranking compounds can be tested for activity
in high-throughput (HTS) functional and cellular screens. HTS
assays for each target RNA can be determined by scintillation
proximity, precipitation, luminescence-based formats, filtration
based assays, colorometric assays, and the like. Lead compounds can
then be scaled up and tested in animal models for activity and
toxicity. The assessment preferably comprises mass spectrometry of
a mixture of the nucleic acid and at least one of the compounds or
a functional bioassay.
[0350] Certain preferred evaluation techniques employing mass
spectroscopy are disclosed in U.S. patent application Ser. No.
______ filed on even date herewith and assigned to the assignee of
the present application. The foregoing patent application is
incorporated herein by reference in its entirety as exemplary of
certain useful and preferred mass spectrometric techniques for use
herewith. It is to be specifically understood, however, that it is
not essential that these particular mass spectrometric techniques
be employed in order to perform the present invention. Rather, any
evaluative technique may be undertaken so long as the objectives of
the present invention are maintained.
[0351] In some embodiments of the invention, the highest ranking
20% of compounds from the hierarchy generated using the DOCK
program are used to generate a further data set of three
dimensional representations of organic compounds comprising
compounds which are chemically related to the compounds ranking
high in the hierarchy. Although the best fitting compounds are
likely to be in the highest ranking 1%, additional compounds, up to
about 20%, are selected for a second comparison so as to provide
diversity (ring size, chain length, functional groups). This
process insures that small errors in the molecular interaction
sites are not propagated into the compound identification process.
The resulting structure/score data from the highest ranking 20%,
for example, is studied mathematically (clustered) to find trends
or features within the compounds which enhance binding. The
compounds are clustered into different groups. Chemical synthesis
and screening of the compounds, described above, allows the
computed DOCK scores to be correlated with the actual binding data.
After the compounds have been prepared and screened, the predicted
binding energy and the observed Kd values are correlated for each
compound.
[0352] The results are used to develop a predictive scoring scheme,
which weighs various factors (steric, electrostatic) appropriately.
The above strategy allows rapid evaluation of a number of scaffolds
with varying sizes and shapes of different functional groups for
the high ranked compounds. In this manner, a further data set of
representations of organic compounds comprising compounds which are
chemically related to the organic compounds which rank high in the
hierarchy can be compared to the numerical representations of the
molecular interaction site to determine a further hierarchy ranked
in accordance with the ability of the organic compounds to form
physical interactions with the molecular interaction site. In this
manner, the further data set of representations of the three
dimensional structures of compound which are related to the
compounds ranked high in the hierarchy are produced and have, in
effect, been optimized by correlating actual binding with virtual
binding. The entire cycle can be iterated as desired until the
desired number of compounds highest in the hierarchy are
produced.
[0353] Compounds which have been determined to have affinity and
specificity for a target biomolecule, especially a target RNA or
which otherwise have been shown to be able to bind to the target
RNA to effect modulation thereof, can, in accordance with preferred
embodiments of this invention, be tagged or labelled in a
detectable fashion. Such labelling may include all of the labelling
forms known to persons of skill in the art such as fluorophore,
radiolabel, enzymatic label and many other forms. Such labelling or
tagging facilitates detection of molecular interaction sites and
permits facile mapping of chromosomes and other useful
processes.
[0354] Summary of Exemplary Mass Spectrometric Techniques
[0355] Mass spectrometry (MS) is a powerful analytical tool for the
study of molecular structure and interaction between small and
large molecules. The current state-of-the-art in MS is such that
less than femtomole quantities of material can be readily analyzed
using mass spectrometry to afford information about the molecular
contents of the sample. An accurate assessment of the molecular
weight of the material may be quickly obtained, irrespective of
whether the sample's molecular weight is several hundred, or in
excess of a hundred thousand, atomic mass units or Daltons (Da). It
has now been found that mass spectrometry can elucidate significant
aspects of important biological molecules. One reason for the
utility of MS as an analytical tool in accordance with the
invention is the availability of a variety of different MS methods,
instruments, and techniques which can provide different pieces of
information about the samples.
[0356] One such MS technique is electrospray ionization mass
spectrometry (ESI-MS) (Smith et al., Anal. Chem., 1990, 62,
882-899; Snyder, in Biochemical and biotechnological applications
of electrospray ionization mass, American Chemical Society,
Washington, D.C., 1996; Cole, in Electrospray ionization mass
spectrometry: fundamentals, instrumentation, Wiley, New York,
1997). ESI produces highly charged droplets of the sample being
studied by gently nebulizing the sample solution in the presence of
a very strong electrostatic field. This results in the generation
of highly charged droplets that shrink due to evaporation of the
neutral solvent and ultimately lead to a "Coulombic explosion" that
affords multiply charged ions of the sample material, typically via
proton addition or abstraction, under mild conditions. ESI-MS is
particularly useful for very high molecular weight biopolymers such
as proteins and nucleic acids greater than 10 kDa in mass, for it
affords a distribution of multiply-charged molecules of the sample
biopolymer without causing any significant amount of fragmentation.
The fact that several peaks are observed from one sample, due to
the formation of ions with different charges, contributes to the
accuracy of ESI-MS when determining the molecular weight of the
biopolymer because each observed peak provides an independent means
for calculation of the molecular weight of the sample. Averaging
the multiple readings of molecular weight so obtained from a single
ESI-mass spectrum affords an estimate of molecular weight that is
much more precise than would be obtained if a single molecular ion
peak were to be provided by the mass spectrometer. Further adding
to the flexibility of ESI-MS is the capability to obtain
measurements in either the positive or negative ionization
modes.
[0357] Matrix-Assisted Laser Desorption/Ionization Mass
Spectrometry (MALDI-MS) is another method that can be used for
studying biomolecules (Hillenkamp et al., Anal. Chem., 1991, 63,
1193A-1203A). This technique ionizes high molecular weight
biopolymers with minimal concomitant fragmentation of the sample
material. This is typically accomplished via the incorporation of
the sample to be analyzed into a matrix that absorbs radiation from
an incident UV or IR laser. This energy is then transferred from
the matrix to the sample resulting in desorption of the sample into
the gas phase with subsequent ionization and minimal fragmentation.
One of the advantages of MALDI-MS over ESI-MS is the simplicity of
the spectra obtained as MALDI spectra are generally dominated by
singly charged species. Typically, the detection of the gaseous
ions generated by MALDI techniques, are detected and analyzed by
determining the time-of-flight (TO) of these ions. While MALDI-TOF
MS is not a high resolution technique, resolution can be improved
by making modifications to such systems, by the use of tandem MS
techniques, or by the use of other types of analyzers, such as
Fourier transform (FT) and quadrupole ion traps.
[0358] Fourier transform mass spectrometry (FTMS) is an especially
useful analytical technique because of its ability to make mass
measurements with a combination of accuracy and resolution that is
superior to other MS detection techniques, in connection with ESI
or MALDI ionization (Amster, J. Mass Spectrom., 1996, 31,
1325-1337). Further it may be used to obtain high resolution mass
spectra of ions generated by any of the other ionization
techniques. The basis for FTMS is ion cyclotron motion, which is
the result of the interaction of an ion with a unidirectional
magnetic field. The mass-to-charge ratio of an ion (m/q or m/z) is
determined by a FTMS instrument by measuring the cyclotron
frequency of the ion. The insensitivity of the cyclotron frequency
to the kinetic energy of an ion is one of the fundamental reasons
for the very high resolution achievable with FTMS. FTMS is an
excellent detector in conventional or tandem mass spectrometry, for
the analysis of ions generated by a variety of different ionization
methods including ESI and MALDI, or product ions resulting from
collisionally activated dissociation (CAD).
[0359] Collisionally activated dissociation (CAD), also known as
collision induced dissociation (CID), is a method by which analyte
ions are dissociated by energetic collisions with neutral or
charged species, resulting in fragment ions which can be
subsequently mass analyzed. Mass analysis of fragment ions from a
selected parent ion can provide certain sequence or other
structural information relating to the parent ion. Such methods are
generally referred to as tandem mass spectrometry (MS or MS/MS)
methods and are the basis of the some of MS based biomolecular
sequencing schemes being employed today.
[0360] FTICR-MS, like ion trap and quadrupole mass analyzers,
allows selection of an ion that may actually be a weak non-covalent
complex of a large biomolecule with another molecule (Marshall and
Grosshans, Anal. Chem., 1991, 63, A215-A229; Beu et al., J. Am.
Soc. Mass Spectrom., 1993, 4, 566-577; Winger et al., J. Am. Soc.
Mass Spectrom., 1993, 4, 566-577); (Huang and Henion, Anal. Chem.,
1991, 63, 732-739), or hyphenated techniques such as LC-MS (Bruins,
Covey and Henion, Anal. Chem., 1987, 59, 2642-2646 Huang and
Henion, J. Am. Soc. Mass Spectrom., 1990, 1, 158-65; Huang and
Henion, Anal. Chem., 1991, 63, 732-739) and CE-MS (Cai and Henion,
J. Chromatogr., 1995, 703, 667-692) experiments. FTICR-MS has also
been applied to the study of ion-molecule reaction pathways and
kinetics.
[0361] So-called "Hyphenated" techniques can be used for structure
elucidation because they provide the dual features of separation
and mass detection. Such techniques have been used for the
separation and identification of certain components of mixtures of
compounds such as those isolated from natural products, synthetic
reactions, or combinatorial chemistry. Hyphenated techniques
typically use a separation method as the first step; liquid
chromatography methods such as HPLC, microbore LC, microcapillary
LC, or capillary electrophoresis are typical separation methods
used to separate the components of such mixtures. Many of these
separation methods are rapid and offer high resolution of
components while also operating at low flow rates that are
compatible with MS detection. In those cases where flow rates are
higher, the use of `megaflow` ESI sources and sample splitting
techniques have facilitated their implementation with on-line mass
spectrometry. The second stage of these hyphenated analytical
techniques involves the injection of separated components directly
into a mass spectrometer, so that the spectrometer serves as a
detector that provides information about the mass and composition
of the materials separated in the first stage. While these
techniques are valuable from the standpoint of gaining an
understanding of the masses of the various components of
multicomponent samples, they are incapable of providing structural
detail. Some structural detail, however, may be ascertained through
the use of tandem mass spectrometry, e.g., hydrogen/deuterium
exchange or collision induced disassociation.
[0362] Typically, tandem mass spectrometry (MS.sup.n) involves the
coupled use of two or more stages of mass analysis where both the
separation and detection steps are based on mass spectrometry. The
first stage is used to select an ion or component of a sample from
which further structural information is to be obtained. This
selected ion is then fragmented by (CID) or photodissociation. The
second stage of mass analysis is then used to detect and measure
the mass of the resulting fragments or product ions. The advent of
FTICR-MS has made a significant impact on the utility of tandem,
MS.sup.n procedures because of the ability of FTICR to select and
trap specific ions of interest and its high resolution and
sensitivity when detecting fragment ions. Such ion selection
followed by fragmentation routines can be performed multiple times
so as to essentially completely dissect the molecular structure of
a sample. A two-stage tandem MS experiment would be called a MS-MS
experiment while an n-stage tandem MS experiment would be referred
to as a MS.sup.n experiment. Depending on the complexity of the
sample and the level of structural detail desired, MS.sup.n
experiments at values of n greater than 2 may be performed.
[0363] Ion trap-based mass spectrometers are particularly well
suited for such tandem experiments because the dissociation and
measurement steps are temporarily rather than spatially separated.
For example, a common platform on which tandem mass spectrometry is
performed is a triple quadrupole mass spectrometer. The first and
third quadrupoles serve as mass filters while the second quadrupole
serves as a collision cell for CAD. In a trap based mass
spectrometer, parent ion selection and dissociation take place in
the same part of the vacuum chamber and are effected by control of
the radio frequency wavelengths applied to the trapping elements
and the collision gas pressure. Hence, while a triple quadrupole
mass analyzer is limited to two stages of mass spectrometry (i.e.
MS/MS), ion trap-based mass spectrometers can perform MS.sup.n
analysis in which the parent ion is isolated, dissociated, mass
analyzed and a fragment ion of interest is isolated, further
dissociated, and mass analyzed and so on. A number of MS.sup.4
procedures and higher have appeared in the literature in recent
years and can be used here. (Cheng et al., Techniques in Protein
Chemistry, VII, pp. 13-21).
[0364] ESI and MALDI techniques have found application for the
rapid and straightforward determination of the molecular weight of
certain biomolecules (Feng and Konishi, Anal. Chem., 1992, 64,
2090-2095; Nelson, Dogruel and Williams, Rapid Commun. Mass
Spectrom., 1994, 8, 627-631). These techniques have been used to
confirm the identity and integrity of certain biomolecules such as
peptides, proteins, oligonucleotides, nucleic acids, glycoproteins,
oligosaccharides and carbohydrates. Further, these MS techniques
have found biochemical applications in the detection and
identification of post-translational modifications on proteins.
Verification of DNA and RNA sequences that are less than 100 bases
in length has also been accomplished using ESI with FTMS to measure
the molecular weight of the nucleic acids (Little et al, Proc.
Natl. Acad. Sci. USA, 1995, 92, 2318-2322).
[0365] ESI tandem MS has been used for the study of high molecular
weight proteins, for peptide and protein sequencing, identification
of post-translational modifications such as phosphorylation,
sulfation or glycosylation, and for the study of enzyme mechanisms
(Rossomando et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 5779-578;
Knight et al., Biochemistry, 1993, 32, 2031-2035). Covalent
enzyme-intermediate or enzyme-inhibitor complexes have been
detected using ESI and analyzed by ESI-MS to ascertain the site(s)
of modification on the enzyme. The literature has shown examples of
protein sequencing where the multiply charged ions of the intact
protein are subjected to collisionally activated dissociation to
afford sequence informative fragment ions (Light-Wahl et al., Biol.
Mass Spectrom., 1993, 22, 112-120). ESI tandem MS has also been
applied to the study of oligonucleotides and nucleic acids (Ni et
al., Anal. Chem., 1996, 68, 1989-1999; Little, Thannhauser and
McLafferty, Proc. Natl. Acad. Sci., 1995, 92, 2318-2322).
[0366] While tandem ESI mass spectra of oligonucleotides are often
complex, several groups have successfully applied ESI tandem MS to
the sequencing of large oligonucleotides (McLuckey, Van Berkel and
Glish, J. Am. Soc. Mass Spectrom., 1992, 3, 60-70; McLuckey and
Habibigoudarzi, J. Am. Chem. Soc., 1993, 115, 12085-12095; Little
et al., J. Am. Chem. Soc., 1994, 116, 4893-4897). General rules for
the principal dissociation pathways of oligonucleotides, as
formulated by McLuckey (McLuckey, Van Berkel and Glish, J. Am. Soc.
Mass Spectrom., 1992, 3, 60-70; Mcluckey and Habibigoudarzi, J. Am.
Chem. Soc., 1993, 115, 12085-12095), have assisted interpretation
of mass spectra of oligonucleotides, and include observations of
fragmentation such as, for example, the stepwise loss of base
followed by cleavage of the 3'-C--O bond of the relevant sugar.
Besides the use of ESI with tandem MS for oligonucleotide
sequencing, two other mass spectrometric methods are also
available: mass analysis of products of enzymatic cleavage of
oligonucleotides (Pieles et al., Nucleic Acids Res., 1993, 21,
3191-3196; Shaler et al., Rapid Commun. Mass Spectrom., 1995, 9,
942-947; Glover et al., Rapid Commun. Mass Spectrom., 1995, 9,
897-901), and the mass analysis of fragment ions arising from the
initial ionization/desorption event, without the use of mass
selection techniques (Little et al., Anal. Chem., 1994, 66,
2809-2815; Nordhoff et al., J. Mass Spectrom., 1995, 30, 99-112;
Little et al., J. Am. Chem. Soc., 1994, 116, 4893-4897; Little and
McLafferty, J. Am. Chem. Soc., 1995, 117, 6783-6784). While
determining the sequence of deoxyribonucleic acids (DNA) is
possible using ESI-MS and CID techniques (McLuckey, Van Berkel and
Glish, J. Am. Soc. Mass Spectrom., 1992, 3, 60-70; McLuckey and
Habibigoudarzi, J. Am. Chem. Soc., 1993, 115, 12085-12095), the
determination of RNA sequence is much more difficult. Thus while
small RNA, such as 6-mers, have been sequenced (McCloskey et al.,
J. Am. Chem. Soc., 1993, 115, 12085-1095), larger RNA have been
difficult to sequence using mass spectrometry.
[0367] Electrospray mass spectrometry has been used to study
biochemical interactions of biopolymers such as enzymes, proteins
and nucleic acids with their ligands, receptors, substrates or
inhibitors. While interactions that lead to covalent modification
of the biopolymer have been studied for some time, those
interactions that are of a non-covalent nature have been
particularly difficult to study heretofore by methods other than
kinetic techniques. It is now possible to yield information on the
stoichiometry and nature of such non-covalent interactions from
mass spectrometry. MS can provide information about the
interactions between biopolymers and other molecules in the gas
phase; however, experiments have demonstrated that the data so
generated can be reflective of the solution phase phenomena from
which the mass spectra were generated.
[0368] ESI is a gentle ionization method that results in no
significant molecular fragmentation and preserves even weakly bound
complexes between biopolymers and other molecules so that they are
detected intact with mass spectrometry. A variety of non-covalent
complexes of biomolecules have been studied using ESI-MS and
reported in the literature (Loo, Bioconjugate Chemistry, 1995, 6,
644-665; Smith et al., J. Biol. Mass Spectrom. 1993, 22, 493-501;
Li et al., J. Am. Chem. Soc., 1993, 115, 8409-8413). These include
the peptide-protein complexes (Busman et al., Rapid Commun. Mass
Spectrom., 1994, 8, 211-216; Loo, Holsworth and Root-Bernstein,
Biol. Mass Spectrom., 1994, 23, 6-12; Anderegg and Wagner, J. Am.
Chem. Soc., 1995, 117, 1374-1377; Baczynskyj, Bronson and Kubiak.,
Rapid Commun. Mass Spectrom., 1994, 8, 280-286), interactions of
polypeptides and metals (Loo, Hu and Smith, J. Am. Soc. Mass
Spectrom., 1994, 5, 959-965; Hu and Loo, J. Mass Spectrom., 1995,
30, 1076-1079; Witkowska et al., J. Am. Chem. Soc., 1995, 117,
3319-3324; Lane et al., J. Cell Biol., 1994, 125, 929-943),
protein-small molecule complexes (Ganem and Henion, ChemTracts-Org.
Chem., 1993, 6, 1-22; Henion et al., Ther. Drug Monit., 1993, 15,
563-569; Baca and Kent, J. Am. Chem. Soc., 1992, 114, 3992-3993),
the study of the quaternary structure of multimeric proteins (Baca
and Kent, J. Am. Chem. Soc., 1992, 114, 3992-3993; Light-Wahl,
Schwartz and Smith, J. Am. Chem. Soc., 1994, 116, 5271-5278; Loo,
J. Mass Spectrom., 1995, 30, 180-183), and the study of nucleic
acid complexes (Light-Wahl et al., J. Am. Chem. Soc., 1993, 115,
803-804; Gale et al., J. Am. Chem. Soc., 1994, 116, 6027-6028;
Goodlett et al., Biol. Mass Spectrom., 1993, 22, 181-183; Ganem, Li
and Henion, Tet. Lett., 1993, 34, 1445-1448; Doctycz et al., Anal.
Chem., 1994, 66, 3416-3422; Bayer et al., Anal. Chem., 1994, 66,
3858-3863; Greig et al., J. Am. Chem. Soc., 1995, 117,
10765-766).
[0369] While data generated and conclusions reached from ESI-MS
studies for weak non-covalent interactions generally reflect, to
some extent, the nature of the interaction found in the
solution-phase, it has been pointed out in the literature that
control experiments are necessary to rule out the possibility of
ubiquitous non-specific interactions (Smith and Light-Wahl, Biol.
Mass Spectrom., 1993, 22, 493-501). Some have applied the use of
ESI-MS and MALDI-MS to the study of multimeric proteins for the
gentleness of the electrospray/desorption process allows weakly
bound complexes, held together by hydrogen bonding, hydrophobic
and/or ionic interactions, to remain intact upon transfer to the
gas phase. The literature shows that not only do ESI-MS data from
gas-phase studies reflect the non-covalent interactions found in
solution, but that the strength of such interactions may also be
determined. The binding constants for the interaction of various
peptide inhibitors to src SH2 domain protein, as determined by
ESI-MS, were found to be consistent with their measured solution
phase binding constants (Loo, Hu and Thanabal, Proc. 43.sup.rd ASMS
Conf. on Mass Spectrom. and Allied Topics, 1995). ESI-MS has also
been used to generate Scatchard plots for measuring the binding
constants of vancomycin antibiotics with tripeptide ligands (Lim et
al., J. Mass Spectrom., 1995, 30, 708-714).
[0370] Similar experiments have been performed to study
non-covalent interactions of nucleic acids. Both ESI-MS and
MALDI-MS have been applied to study the non-covalent interactions
of nucleic acids and proteins. While MALDI does not typically allow
for survival of an intact non-covalent complex, the use of
crosslinking methods to generate covalent bonds between the
components of the complex allows for its use in such studies.
Stoichiometry of interaction and the sites of interaction have been
ascertained for nucleic acid-protein interactions (Jensen et al.,
Rapid Commun. Mass Spectrom., 1993, 7, 496-501; Jensen et al., 42nd
ASMS Conf. on Mass Spectrom. and Allied Topics, 1994, 923). The
sites of interaction are typically determined by proteolysis of
either the non-covalent or covalently crosslinked complex (Jensen
et al., Rapid Commun. Mass Spectrom., 1993, 7, 496-501; Jensen et
al., 42nd ASMS Conf. on Mass Spectrom. and Allied Topics, 1994,
923; Cohen et al., Protein Sci., 1995, 4, 1088-1099). Comparison of
the mass spectra with those generated from proteolysis of the
protein alone provides information about cleavage site
accessibility or protection in the nucleic acid-protein complex
and, therefore, information about the portions of these biopolymers
that interact in the complex.
[0371] Electrospray mass spectrometry has also been effectively
used for the determination of binding constants of noncovalent
macromolecular complexes such as those between proteins and
ligands, enzymes and inhibitors, and proteins and nucleic acids.
Greig et al. (J. Am. Chem. Soc., 1995, 117, 10765-10766) have
reported the use of ESI-MS to determine the dissociation constants
(K.sub.D) for oligonucleotide-bovine serum albumin (BSA) complexes.
The K.sub.D values determined by ESI-MS were reported to match
solution K.sub.D values obtained using capillary
electrophoresis.
[0372] Cheng et al. (J. Am. Chem. Soc., 1995, 117, 8859-8860) have
reported the use of ESI-FTICR mass spectrometry as a method to
determine the structure and relative binding constants for a
mixture of competitive inhibitors of the enzyme carbonic anhydrase.
Using a single ESI-FTICR-MS experiment these researchers were able
to ascertain the relative binding constants for the noncovalent
interactions between inhibitors and the enzyme by measuring the
relative abundances of the ions of these noncovalent complexes.
Further, the K.sub.Ds so determined for these compounds paralleled
their known binding constants in solution. The method was also
capable of identifying the structures of tight binding ligands from
small mixtures of inhibitors based on the high resolution
capabilities and multistep dissociation mass spectrometry afforded
by the FTICR technique. In a related study, Gao et al. (J. Med.
Chem., 1996, 39, 1949-55) have reported the use of ESI-FTICR-MS to
screen libraries of soluble peptides in a search for tight binding
inhibitors of carbonic anhydrase II. Simultaneous identification of
the structure of a tight binding peptide inhibitor and
determination of its binding constant was performed. The binding
affinities determined from mass spectral ion abundance were found
to correlate well with those determined in solution experiments.
Further, the applicability of this technique to drug discovery
efforts is limited by the lack of information generated with
regards to sites and mode of such noncovalent interactions between
a protein and ligands.
[0373] Also, these methods discuss, and appear to be limited to,
the study of ligand interactions with proteins. The suitability of
this method of mass spectrometric analysis of binding and
dissociation constants for the study of noncovalent interactions of
oligonucleotides, nucleic acids, such as RNA and DNA, and other
biopolymers has not been described in the literature.
[0374] The drug discovery process has recently been revolutionized
by the introduction of high throughput synthesis and combinatorial
chemistry which afford collections and mixtures of large numbers of
synthetic compounds for the purpose of screening for biological
activity. Such large mixtures and pools of compounds pose
significant challenges for the bioassay and analytical scientist.
The analytical challenge is two-fold: separation of the active
component of a mixture, and the identification of its structure. A
variety of separation methods are available, including LC, HPLC,
and CE. However, from the standpoint of separating biologically
active components from a mixture of one or more targets with a
combinatorial library necessitates the use and development of
methods that select for and separate the complex (usually
noncovalent) between the ligands and the target. Affinity column
methods have been used to selectively isolate and subsequently
analyze binding components of mixtures of compounds. For example,
Kassel et al. (Kassel et al., Techniques in Protein Chemistry VI,
J. Crabb, Ed., Academic Press, San Diego, 1995, 39-46) have used an
immobilized src SH2 domain protein column to separate and then
analyze by HPLC-ESI-MS the structure of high affinity binding
phosphopeptides.
[0375] A similar technique, ACE-ESI-MS, uses affinity capillary
electrophoresis to accomplish the separation of noncovalent
complexes formed upon mixing a biomolecular target with a
combinatorial library or mixture of compounds. The receptor is
typically incorporated into the capillary so that those ligands
present in the combinatorial mixture interact with the target and
are retained or slowed down within the capillary. Once separated,
these noncovalent complexes are analyzed on-line by ESI-MS to
ascertain the structures of the complexes and bound components.
This method incorporates into one, the two steps that were
previously performed separately: the compound/noncovalent complex
selection, as has previously been demonstrated for vancomycin (Chu
et al., Acc. Chem. Res., 1995, 28, 461-468; Chu et al., J. Org.
Chem., 1993, 58, 648-52) and the step of compound identification
(Cai and Henion, J. Chromatogr., 1995, 703, 667-692). For example,
ACE-ESI-MS has been applied to mixtures of vancomycin with peptide
libraries (Chu et al., J. Am. Chem. Soc., 1996, 118, 7827-35) to
allow rapid screening of noncovalent complexes formed, and the
identification of peptides that bind to vancomycin. Another method
for the separation and identification of active components from
combinatorial libraries is the use of size-exclusion chromatography
(SEC) followed by LC/MS or CE/MS analysis. Size exclusion is a
simple yet powerful method to separate a biopolymer target and its
complexes with small molecules members of a combinatorial library.
Once isolated by SEC, these complexes are dissociated, under
denaturing solution conditions, and finally the binding ligands are
analyzed by mass spectrometry. This method has been applied to the
identification of high affinity ligands for human serum albumin
(HSA) from combinatorial library of small molecules (Dunayevskiy et
al., Rapid Commun. Mass Spectrom., 1997, 11, 1178-84).
[0376] Bio-affinity characterization mass spectrometry (BACMS) is
yet another method for the characterization of noncovalent
interactions of mixtures of ligands and biomolecular targets (Bruce
et al., Rapid Commun. Mass Spectrom., 1995, 9, 644-50). BACMS
involves the electrospray ionization of a solution containing both
the affinity target and a mixture of ligands (or a combinatorial
library), followed by trapping of all the ionic species in the
FTICR ion-trap. The complexes of interest are then identified in
the mass spectrum and isolated by selected-ion accumulation. This
is followed by low energy dissociation or `heating` to separate the
high binding affinity ligands present in the complex. Finally,
collisionally activated dissociation (CAD) is used to provide
structural information about the high binding affinity ligand. The
greatest advantage of BACMS is that the time-consuming techniques
usually needed for the study of libraries, such as affinity
chromatography, using solid supports for separation and
purification of the complexes, followed by analysis to characterize
the selected ligands, are all combined into one FTICR-MS
experiment. To date, BACMS has only been applied to the study of
protein targets.
[0377] None of the foregoing methods, however, have demonstrated
applicability to a variety of biomolecular targets. Further, such
methods do not provide rapid determination of the site of
interaction between a combinatorially derived ligand and
biopolymer.
[0378] Tandem mass spectrometry, as performed using electrospray
ionization (ESI) on FTICR, triple quadrupole, or ion-trap mass
spectrometers, has been found to be a powerful tool for determining
the structure of biomolecules. It is known in the art that both
small and large (>3000 kbase) RNA and DNA may be transferred
from solution into the gas phase as intact ions using electrospray
techniques. Further it is known, to those skilled in the art that
these ions retain some degree of their solution structures as ions
in the gas phase; this is especially useful when studying
noncovalent complexes of nucleic acids and proteins, and nucleic
acids and small molecules by mass spectrometric techniques.
[0379] Summary of Certain Preferred Embodiments
[0380] Studies have demonstrated that oligonucleotides and nucleic
acids obey certain fragmentation patterns during collisionally
induced dissociation (CID), and that these fragments and patterns
can be used to determine the sequence of the nucleic McLuckey, Van
Berkel and Glish, J. Am. Soc. Mass Spectrom., 1992, 3, 60-70;
Mcluckey and Habibigoudarzi, J. Am. Chem. Soc., 1993, 115,
12085-12095). Electrospray ionization produces several multiply
charged ions of the parent nucleic acid, without any significant
fragmentation of the nucleic acid. Typically, a single charge state
of the nucleic acid is isolated using a triple quadrupole ion trap,
or ion cyclotron resonance (ICR) device. This ion is then excited
and allowed to collide with a neutral gas such as helium, argon or
nitrogen so as to afford cleavage of certain bonds in the nucleic
acid ion, or excited and fragmented with a laser pulse. Typically,
two series of fragment ions are found to be formed: the a-Base
series, and the w-series.
[0381] The series of a-Base fragments originates from initial
cleavage of the glycosidic bond by simultaneous abstraction of a
C-2' proton, followed by the elimination of the 3'-phosphate group
and the C-4' proton. This fragmentation scheme results in a
residual furan attached to the 3'-phosphate and affords a series of
a-Base fragments whose masses increase sequentially from the
5'-terminus of the nucleic acid. Measurement of the masses of these
collisionally induced fragments therefore affords the determination
of the sequence of the nucleic acid in the 5' to 3' direction. The
w series of fragments is generated via cleavage of the nucleic acid
in a manner that leaves a 5'phosphate residue on each fragment.
Thus monitoring the masses of w-series fragments allows
determination of the sequence of the nucleic acid in the 3' to 5'
direction. Using the sequence information generated from both
series of fragments the sequence of deoxyribonucleic acids (DNA)
may be ascertained. Obtaining similar mass spectrometric
information for ribonucleic acids (RNA), is a much more difficult
task. Collisionally induced dissociation (CID) of RNA is much less
energetically favored than is the case for DNA because of the
greater strength of the glycosidic bond in RNA. Hence, while small
RNA such as 6-mers have been sequenced using CID MS, the sequencing
of larger RNA has not been generally successful using tandem
MS.
[0382] Determination of the structure of biomolecules, such as
proteins and nucleic acids, may be attempted using solution
biochemical cleavage followed by mass spectrometry. However, these
methods are cumbersome and not always successful in that several
biochemical cleavage and separation steps need to be performed
prior to MS analysis of the cleaved products. Also, the level of
information provided with regards to secondary and tertiary
structure of biomolecules is limited. Methods available in the
scientific literature are therefore greatly limited in terms of the
sequence and structural information they provide for biomolecules
and biomolecular targets.
[0383] One aspect of the present invention provides methods for
determining the structure of biomolecular targets such as nucleic
acids using mass spectrometry. The structure of nucleic acids,
especially RNA, which is often difficult to ascertain, is readily
determined using the methods of this invention. The structure of a
nucleic acid is determined from the fragmentation pattern observed
in MS.sup.n experiments. Directed fragmentation of RNA is
facilitated by the selective incorporation of deoxynucleotides or
other nucleosidic residues at specific residue locations in the
nucleic acid sequence. During CID of such RNA/DNA chimeric nucleic
acids, cleavage is facilitated at the sites where deoxynucleotides
or the other non-native residues were incorporated. Cleavage is
also influenced by the local secondary and tertiary structure of
the biomolecule. Therefore, the cleavage patterns observed from a
RNA/DNA hybrid reveals the local structure of the nucleic acid,
including mismatched base pairs, bulged regions and other
features.
[0384] Since exposed deoxynucleotide residues are known to be
susceptible to CID cleavage in MS experiments, the systematic
incorporation of such residues into RNA allows the systematic
exploration of the local structure of RNA. Using this embodiment of
the invention, it is possible to determine the secondary and
tertiary structure of nucleic acids, including features such as
mismatched base pairs, loops, bulges, and kink and stem
structures.
[0385] Determination of the structure of an RNA may be
accomplished, using exemplary methods of the invention, as follows.
An RNA whose structure is to be determined is synthesized using an
automated nucleic acid synthesizer. During RNA synthesis,
deoxynucleotides are selectively incorporated into the sequence at
specific sites where the structure is to be probed. This RNA/DNA
chimeric nucleic acid, which is sensitized to collisional
activation, is now used for sequence and structure determination
using tandem MS experiments. ESI-MS, followed by trapping of
selected ions and subsequent CID of each ion, affords information
as to which positions of the nucleic acid hybrid are disordered (or
not participating in a higher order structure) and, therefore,
available for cleavage. A systematic pattern of deoxynucleotide
incorporation into the sequence of the test RNA allows a
systematic, mass spectrometric assessment of structure in a certain
area of the nucleic acid, or for the entire nucleic acid. Other
modified nucleic acid residues may be used instead of DNA. This,
chemically modified nucleic acid subunits such as Z.sup.1-modified,
e.g. 2.sup.1-OAlky, base-modified, backbone modified or other
residues may serve. Such residues will permit assessment of DNA as
well as RNA.
[0386] The present invention also provides methods for the
determination of the site and nature of interactions between a
biomolecular target and a binding ligand. This is information of
critical value to the process of drug discovery. Current methods of
biomolecular screening do not provide a straightforward means of
also determining the nature of the interaction between a binding
ligand and the biomolecular target. Information such as the
stoichiometry and binding affinity of the interaction often needs
to be ascertained from additional biochemical assays, thus slowing
down and increasing the cost of drug discovery. It is often the
case that binding of a drug or ligand to a biomolecular target,
such as a nucleic acid, may lead to a change in conformation of the
biomolecule to a different structure. This, too, may contribute to
protection of the biomolecule from cleavage.
[0387] The present invention provides convenient methods for
determining the site or sites on a biomolecular target where a
binding ligand interacts. This is accomplished based on the
knowledge that collisionally activated dissociation (CID or CAD) of
a noncovalent biomolecule-ligand complex may be performed such that
cleavage of the complex occurs only at exposed sites of the
biomolecules. Thus cleavage sites present on the biomolecule that
are involved in binding with the ligand are protected because of
the increased structural order from the binding event during CID.
ESI-MS.sup.n spectra generated using this method, in the presence
and absence of a binding ligand (or drug), will reveal differential
fragmentation patterns due to ligand induced protection of cleavage
sites. Comparison of the mass spectra generated in the presence and
absence of a binding ligand will, therefore, reveal the positions
in the biomolecular sequence where the interactions between ligand
and biomolecule are occurring.
[0388] These methods for determining the sites of interaction
between a binding ligand and a biomolecular target are broadly
applicable. The biomolecular targets that may be studied using this
method include, but are not limited to, peptides, proteins,
antibodies, oligonucleotides, RNA, DNA, other nucleic acids,
glycopeptides, and oligosaccharides. It is preferred that the
biomolecular target be a nucleic acid. It is further preferred that
the biomolecular target be a chimeric RNA/DNA nucleic acid,
synthesized to selectively incorporate deoxynucleotides, (or other
residues) in the sequence at specific locations. The binding ligand
may be one of the groups of molecules including, but not limited
to, organic or inorganic, small to large molecular weight
individual compounds, mixtures and combinatorial libraries of
ligands, inhibitors, agonists, antagonists, substrates, and
biopolymers, such as peptides or oligonucleotides.
[0389] Determination of the sites on an RNA target where
interaction occurs with a binding ligand may be accomplished as
follows. An RNA target that is to be studied as a biomolecular
target is prepared using an automated synthesizer, and selectively
incorporating deoxynucleotides into the sequence at specific sites.
An aliquot of this RNA/DNA chimeric is used directly for ESI-MS,
followed by CID analysis of selectively accumulated ions, to
establish the native structure and cleavage patterns of this
biomolecular target. A second aliquot of the RNA/DNA chimeric is
mixed with a solution of a drug or ligand that is known to bind to
the biomolecular target. The target and ligand are anticipated to
interact in solution to form a noncovalent complex. Subjecting this
solution of the noncovalent biomolecule-ligand complex to the
method of this invention leads to ionization of the complex with a
retention of the noncovalent interactions and binding
stoichiometries. CID of the complex then leads to cleavage of the
biomolecule sequence at fragmentation sites that are exposed. Sites
where fragmentation would otherwise occur, but which are involved
in binding the ligand to the biomolecule, are protected, such that
cleavage at or near these sites is prevented during the CID stage.
The differences in the fragmentation patterns of the biomolecule
when subjected to the methods of this invention in the presence and
absence of binding ligand indicate the site(s) on the biomolecule
that is protected and, therefore, are involved in binding the
ligand.
[0390] Likewise, a systematic pattern of deoxynucleotide
incorporation into the sequence of the test RNA will allow for a
systematic mass spectrometric assessment of binding sites and
interactions in a certain area of the nucleic acid, or for the
entire nucleic acid, using the method of this invention. This
invention, therefore, also provides a new method of `footprinting`
biomolecular targets especially nucleic acids. This footprinting by
mass spectrometry is a straightforward method for mapping the
structure of biomolecular targets and the sites of interactions of
ligands with these targets.
[0391] The nature of interactions between the binding ligand and a
biomolecular target are also readily studied using the method of
this invention. Thus, the stoichiometry and relative dissociation
constant of the biomolecule-ligand noncovalent complex is readily
ascertained using the method of this invention. The ratio of the
number of ligand molecules and the number of biomolecular receptors
involved in the formation of a noncovalent biomolecule-ligand
complex is of significant importance to the biochemist and
medicinal chemist. Likewise, the strength of a noncovalent complex,
or the binding affinity of the ligand for the biomolecular target,
is of significance because it provides an indication of the degree
of complementarity between the ligand and the biomolecule. Also,
the determination of this binding affinity is important for the
rank ordering of different ligands so as to provide
structure-activity relationships for a series of ligands, and to
facilitate the design of stronger binding ligands for a particular
biomolecular target.
[0392] The methods of the present invention are also capable of
determining both the binding stoichiometry and affinity of a ligand
for the biomolecular target being screened in a screening study.
Electrospray ionization is known to retain to a significant degree,
the solution phase structures of biomolecules and their noncovalent
complexes in the gaseous ions it generates. Thus, determination of
the stoichiometry of noncovalent complexes simply needs data on the
masses of the ligand, biomolecular target and the noncovalent
biomolecule-ligand complex. The data needed to accomplish this
determination is actually available from the mass spectrometry
experiment that may be performed to determine the structure and
site of binding of a ligand to the biomolecular target. Based on
the knowledge of the structure and sequence of the target
biomolecule, MS analysis of the biomolecule-ligand complex reveals
the number of ligand and target molecules present in the
noncovalent complex. If the noncovalent complex ion observed from
the mass spectrum is of an m/z equal to that expected from the
addition of the m/z values of one molecule each of the target
biomolecule and ligand, then the noncovalent complex must be formed
from a 1:1 interaction between the biomolecule and ligand. Simple
mathematical operations on the molecular weight and charges of the
target and ligand can likewise determine higher levels of
interactions between ligand and biomolecule. The high resolution of
a FTICR mass spectrometer allows direct identification of the bound
ligand based on exact measurement of the molecular mass of the
complex relative to unbound nucleic acid.
[0393] The use of mass spectrometry, in accordance with this
invention can provide information on not only the mass to charge
ratio of ions generated from a sample, but also the relative
abundance of such ions. Under standardized experimental conditions,
it is therefore possible to compare the abundance of a noncovalent
biomolecule-ligand complex ion with the ion abundance of the
noncovalent complex formed between a biomolecule and a standard
molecule, such as a known substrate or inhibitor. Through this
comparison, binding affinity of the ligand for the biomolecule,
relative to the known binding of a standard molecule, may be
ascertained. Determination of the nature of the interaction of a
ligand with a biomolecular target may be carried out as exemplified
for the binding of a small molecule ligand with a nucleic acid
target. A chimeric RNA/DNA biomolecular target whose binding to a
test ligand is to be studied is first prepared via automated
synthesis protocols. An aliquot of a known concentration of
chimeric nucleic acid is treated with a known concentration and
quantity of a standard compound that is known to bind that nucleic
acid, such as the aminoglycoside paromomycin which is known to bind
to the 16S A-site of RNA. ESI-MS, followed by CID of the
paromomycin-nucleic acid complex, affords a control spectrum for
the interactions and complex. A second aliquot of the chimeric
nucleic acid is next treated with a test ligand using quantities
and concentrations similar to those used for the control
experiment. Application of the method of the invention to this
nucleic acid-ligand noncovalent complex affords a test spectrum
that reveals the nature of the biomolecule-ligand interaction.
Analysis of the noncovalent nucleic acid-ligand complex based on
the known molecular weights of the two components of the complex
allows the determination of the number of nucleic acid molecules
and ligands present in the complex. Further, comparison of the
abundance of the nucleic acid-ligand complex ion with the abundance
of the ion generated from the e.g. paromomycin-nucleic acid complex
(or complex with any other known interacting species) provides a
convenient and direct estimate of the binding affinity of the test
ligand compared to the standard, paromomycin. Since the standard is
well characterized, its solution binding affinity should be known
from other experiments or literature sources. For example,
paromomycin binds to a test 27-mer RNA with a .about.1 .mu.M
affinity. Knowing the binding affinity of the test ligand relative
to paromomycin from the MS experiment, it is now possible to
determine the micromolar binding affinity of the test ligand for
the nucleic acid target being studied. Relative binding affinity
may also be measured by testing a standard compound and test ligand
simultaneously as in a mixture with the target biomolecule, in a
single test assay.
[0394] Another object of the present invention is to provide
general methods for the screening of compounds for drug discovery.
The invention provides methods for the screening of a wide variety
of biomolecular targets that include, but are not limited to,
peptides, proteins, receptors, antibodies, oligonucleotides, RNA,
DNA, RNA/DNA hybrids, nucleic acids, oligosaccharides,
carbohydrates, and glycopeptides. The molecules that may be
screened by using the methods of this invention include, but are
not limited to, organic or inorganic, small to large molecular
weight individual compounds, mixtures and combinatorial libraries
of ligands, inhibitors, agonists, antagonists, substrates, and
biopolymers, such as peptides or oligonucleotides.
[0395] The primary challenge when screening large collections and
mixtures of compounds is not in finding biologically relevant
activities, for this has been demonstrated in many different cases,
but in identifying the active components from such screens, and
often from mixtures and pools of compounds that are found to be
active. One solution that has been practiced by the art-skilled in
high throughput drug discovery is the iterative deconvolution of
mixtures. Deconvolution essentially entails the resynthesis of that
combinatorial pool or mixture that was found to be active in
screening against a target of interest. Resynthesis may result in
the generation of a set of smaller pools or mixtures, or a set of
individual compounds. Rescreening and iterative deconvolution are
performed until the individual compounds that are responsible for
the activity observed in the screens of the parent mixtures are
isolated.
[0396] However, analytical techniques are limited in their ability
to adequately handle the types of mixtures generated in
combinatorial efforts. The similarity of members of combinatorial
mixtures or pools, and the complexity of such mixtures, prohibit
effective analytical assessment until the mixtures have been
deconvoluted into individual compounds, or at the very least into
pools of only a handful of components. While this process of
deconvolution, involving resynthesis, rescreening and analysis, is
very cumbersome and time-consuming, it is also very costly. A
general method that alleviates these problems by rapidly revealing
active mixtures and identifying the active components of such
mixtures is clearly needed to save time and money in the drug
discovery process.
[0397] The present invention solves the need for a method to
rapidly assess the activity of combinatorial mixtures against a
biomolecular target and also identify the structure of the active
components of such mixtures. This is exemplified by the screening
of combinatorial mixtures for binding to a nucleic acid target as
follows. A chimeric RNA/DNA target of known sequence is selected as
the screening target based on biological relevance. This chimeric
nucleic acid target is prepared via automated synthesis. An aliquot
of the nucleic acid is used at a concentration of 10 .mu.M and
treated with e. q. paromomycin acetate at a concentration of 150
nM. A sample of the mixture is analyzed by the method of the
invention to demonstrate binding of the paromomycin by observation
of the paromomycin-nucleic acid complex ion. Next, an aliquot of
this mixture is treated with a DMSO solution of a combinatorial
mixture of compounds such that the final concentration of each
component of the mixture is .about.150 nM. This sample is then
subjected to ESI-MS, and the mass spectrum monitored for the
appearance of new signals that correspond to new nucleic
acid-ligand noncovalent complexes formed with components of the
combinatorial library.
[0398] The relative dissociation constants of these new complexes
are determined by comparing the abundance of these new ions with
the abundance of the paromomycin-nucleic acid complex ion whose
binding affinity for the target is known a priori. Algorithmic
deconvolution of the new complex ions observed, while taking into
account the masses of the target and the components of the
combinatorial library, provides the molecular weights of the
binding ligands present in the observed noncovalent complexes.
Alternatively, the identity of the binding ligand may also be
determined by first isolating the newly observed complex ion using
a triple quadrupole ion-trap or an ion cyclotron resonance device
(ICR) followed by conventional identification by mass spectrometry
fragment analysis. For example, upon isolation, a noncovalent
complex ion is `heated` or dissociated into the constituent ligand
and biomolecule ions. This MS/MS experiment then can be tuned to
study fragmentation of the ligand. This information provides direct
evidence of the structure of the bound ligand. This method of the
present invention, therefore, provides both the identity and
relative binding affinity of members of combinatorial or other
mixtures of compounds that bind to the nucleic acid target.
[0399] Not only does the present invention provide methods for the
determination of the molecular weight and relative binding affinity
of the binding components of a combinatorial or other mixture of
compounds, but it also provides valuable information about the site
of binding on the biomolecular target. Such information permits the
identification of compounds having particular biological activity
and gives rise to useful drugs, veteriary drugs, agricultural
chemicals, industrial chemicals, diagnostics and other useful
compounds. This can also be accomplished as part of the same mass
spectrometric procedure by isolating the newly observed complex
ions using a triple quadrupole ion-trap or an ion cyclotron
resonance device (ICR). For example, upon isolation, a noncovalent
complex ion is collisionally activated to cleave the chimeric
nucleic acid target at exposed deoxynucleotide sites. This MS/MS
procedure, then, can be tuned to study fragmentation of the
biomolecular target.
[0400] Comparison of the cleavage and fragment patterns so obtained
for the nucleic acid component of the noncovalent complex with
patterns obtained for the native chimeric nucleic acid alone
reveals the locations on the nucleic acid that are protected by the
binding of the ligand. This indicates the binding sites for the
ligand on the nucleic acid. Comparison of the cleavage patterns to
those observed from the CID of the standard-nucleic acid complex
ion provide correlations between the sites of binding of the new
ligand and standard. In this fashion, ligands that bind to nucleic
acid targets may be identified such that they compete for the same
binding site on the nucleic acid where the standard binds, or bind
at completely different and new sites on the nucleic acid. Both
these types of observations are of value from a drug discovery
standpoint.
[0401] Drug discovery, using any one of a number of different types
of biomolecular targets attends use of the methods of this
invention which can rapidly screen large combinatorial libraries
and mixtures of compounds for binding activity against a specific
target.
[0402] It is possible that combinatorial libraries and mixtures of
compounds being used for screening may contain components that are
similar in mass because their elemental compositions are similar
while their structures are different, or at the very least,
isomeric or enantiomeric. In such instances, a simple algorithmic
calculation of the molecular weight of a bound ligand will be
insufficient to provide the identity of the ligand for there may be
multiple components of the same molecular mass. The methods of the
invention are also capable of addressing and resolving such
problems of ligand identification. The use of MS/MS experiments to
further fragment the bound ligand, following selective ion
accumulation of the ligand ion from the noncovalent complex, is a
simple technique that provides structural detail of the bound
ligand. This mass and structural information provided by the
methods of this invention is expected to resolve the vast majority
of mass redundancy problems associated with the screening of large
combinatorial libraries and mixtures of compounds.
[0403] In a preferred embodiment, the present invention also
provides method for simultaneously screening multiple biomolecular
targets against combinatorial libraries and mixtures or collections
of compounds. This is a significant advantage of the present
invention over current state-of-the-art techniques in the screening
of compounds for such binding. There is believed to be no prior
technique that allows the simultaneous and rapid screening of
multiple targets, while providing structural detail on the target
and binding ligand at the same time. In addition to providing
methods for the rapid and simultaneous screening of multiple
biomolecular targets, the present invention also provides methods
for determining the structure and nature of binding of both the
target and binding ligand.
[0404] As discussed above, mass spectrometry methods of the present
invention provide a direct means for screening and identifying
those components of combinatorial mixtures that bind to a target
biomolecule in solution. In order to enhance efficiency, it is
preferable to multiplex the screening process by simultaneously
screening multiple targets for binding activity against a
combinatorial library or mixture of compounds. This strategy is
normally limited by the distribution of charge states and the
undesirable mass/charge overlap that will be generated from all
possible noncovalent biomolecule-ligand complexes that could be
formed during such a screening assay. This problem of overlapping
peaks in the mass spectra is further exacerbated if the
biomolecular targets being screened are of similar sequence,
composition, or molecular weight. In such instances it would not be
possible to ascertain in a rapid and simple operation the
composition of biomolecule-ligand complexes because of the
extensive mass redundancy present in the pool of biomolecules being
studied and possible in the combinatorial library being
screened.
[0405] The method of the present invention alleviates the problem
of biomolecular target mass redundancy through the use of special
mass modifying molecular weight tags. These mass modifying tags are
typically uncharged or positively charged groups such as, but not
limited to, alkyl and tetraalkylammonium groups, and polymers such
as, but not limited to, polyethylene glycols (PEG), polypropylene,
polystyrene, cellulose, sephadex, dextrans, cyclodextrins,
peptides, and polyacrylamides. These mass modifying tags may be
selected based on their molecular weight contribution and their
ionic nature. These mass modifying tags may be attached to the
biopolymeric targets at one or more sites including, but not
limited to, the 2'-O--, 3'-terminus, 5'-terminus or along the
sugar-phosphate backbone of nucleic acid targets. Addition of mass
modifying tags to the 5'terminus of synthetic oligonucleotides can
be realized either using conventional phosphoramidite chemistry,
other conventional chemistry or by biochemical or enzymatic means.
Such mass modification of a nucleic acid may be carried out using
conventional, manual or automated techniques. Alternatively,
addition of mass modifying tags may be performed at the 3'-terminus
by the use of appropriately modified polymer or CPG supports for
solid-phase synthesis of nucleic acids. Mass modification at the
3'terminus may also be done by biochemical or enzymatic means. It
is also possible to attach mass modifying tags to the
internucleotide linkages of a nucleic acid. This may be performed
via the use of appropriately modified phosphoramidites, or other
nucleoside building blocks during nucleic acid synthesis or via
post-synthetic modification of the internucleotide linkage.
Further, attachment of mass modifying tags to nucleic acid targets
may also be accomplished via the use of bifunctional linkers at any
functional site on the nucleic acid. Similarly, when working with
other classes of biomolecular targets these mass modifying tags may
likewise be incorporated at one or more positions on the
biomolecule. As will be apparent, inclusion in either target or
ligand of isotopic mass labels may also be useful.
[0406] Thus, similar nucleic acid and other biological targets may
be differentially tagged for rapid mass spectrometric screening by
the methods of this invention. When noncovalent complexes are
observed from this multiplexed screening of multiple nucleic acid
targets with mixtures of small molecular weight combinatorial
libraries, the constituent ligand and biomolecule are readily
identified using conventional mass analyzers such as quadrupole,
ion trap, ICR, magnetic sector, or TOF and followed by MS/MS. This
is because the mass modifying tags make the m/z (mass to charge
ratio) of the signal arising from each target biomolecule-ligand
complex ion of similar charge, distinct in the mass spectrum, and
which results in cleanly separated ion peaks. Mass redundancy and
peak overlap are both avoided by the use of mass modifying
tags.
[0407] The present invention is also highly useful in combination
with other techniques for the identification of ligands which
interact with molecular interaction sites on RNA and other nucleic
acids. Molecular interaction sites attend RNA and are believe to be
highly important in the functioning of such RNA. The nucleotide
sequences of molecular interaction sites are highly conserved, even
among taxonomically diverse species. Moreover, such molecular
interaction sites have specific structures which provide
opportunities for ligand binding. Ascertaining which ligands bind
to such sites as well as determining the relative affinities and
specificities for the binding of each ligand provides lead
compounds for drug discovery, therapeutics, agricultural chemistry,
industrial chemistry and otherwise.
[0408] The present mass spectrometric techniques, especially the
MASS techniques and those which possess similar analytical
robustness and power, are ideally suited for cooperating with drug
and other discovery and identification programs such as those which
determine ligand binding to molecular interaction sites. The
identification of molecular interaction sites in RNA and other
nucleic acids and the determination of hierarchies of molecular
ligands which likely bind to such molecular interaction sites can
be evaluated through the present techniques. Thus, in accordance
with preferred embodiments of the present invention, a hierarchy of
ligands ranked in accordance with their anticipated or calculated
likelihood of binding to a molecular interaction site of an RNA are
actually synthesized. Such synthesis is preferably accomplished in
an automated or robotized fashion, preferably from instruction sets
provided in attendance to the ranked hierarchy of ligands. The
compounds may be prepared in a library or mixture since the present
mass spectrometric methods can evaluate pluralities of compounds
and their complexes with RNA simultaneously.
[0409] After the ligands are synthesized, preferably in library
form, they are contacted with the RNA having the molecular
interaction site of interest. Complexation or binding
(conventionally, non-covalent binding) is permitted to occur. The
complexed RNA ligand library is then analyzed by mass spectrometry.
A principal object of the analysis is preferably the determination
of which ligands bind to the RNA molecular interaction site and,
among those, which ones rank more highly in terms of specificity
and affinity. Accordingly, it is possible to identify from a
mixture or library of compounds, which ones are the most
interactive with a particular molecular interaction site so as to
be able to modulate it. Such compounds can either be used
themselves, or, more likely, be used as lead compounds for
modification into drugs, agricultural chemicals, environmental
chemicals, industrial and food chemicals and otherwise.
[0410] As described above, it is highly desirable to challenge RNAs
having molecular interaction sites with libraries of compounds
which have already been predicted or calculated to be likely to
interact with the interaction sites. It is preferred that such
molecules belong to ranked hierarchies so as to give rise to the
greatest likelihood of finding highly potent modulators of the
target RNA.
[0411] While there are a number of ways to identify compounds
likely to interact with molecular interaction sites of RNA and
other biological molecules, preferred methodologies are described
in U.S. patent applications filed on even date herewith and
assigned to the assignee of this invention. These application bear
U.S. Serial Nos. (Unknown) and have been assigned attorney docket
numbers IBIS-0002, IBIS 0003, IBIS-0004, IBIS-0006 and IBIS-0007.
All of the foregoing applications are incorporated by reference
herein in their entirety.
[0412] One mass spectrometric method which is particularly useful
when combined with the techniques of the foregoing commonly owned
inventions provides the determination of specificity and affinity
of ligands to RNA targets. MASS (multi target affinity/specificity
screening) techniques can provide high throughput screening methods
to analyze the specificity and affinity of ligands to molecular
interaction sites of nucleic acids, especially RNA. MASS employs
high performance electrospray ionization Fourier transform ion
cyclotron resonance mass spectrometry (ESI-FTICR-MS) to a)
determine exact chemical composition of affinity selected ligands
originating from a combinatorial library, b) determine relative
dissociation constants (Kd) of ligands complexed to the target(s),
and c) determine the location of ligand binding. This information
can be gathered from each target(s) or library set in a single
assay in less than 15 minutes. This scheme benefits from two unique
aspects of the ESI-FTICR combination. The "soft" nature of the
electrospray ionization process allows specific noncovalent
complexes to be ionized and transferred into the gas phase intact
where they are amenable to subsequent characterization by mass
spectrometry. The high resolving power afforded by the FTICR
platform facilitates the characterization of complex mixtures
which, when combined with the high mass accuracy inherent to FTICR,
provides unambiguous identification of ligands complexed with the
molecular interaction site or sites of a target or targets.
[0413] Binding site information can be obtained by comparing the
gas phase fragmentation patterns of the free and complexed target
while relative binding constants are derived from the relative
abundance of complexes using a complex with a known Kd as an
internal standard. With knowledge of the specificity and affinity
of ligands to the molecular interaction site of a target RNA, the
desired lead or ultimate compound for modulation of the RNA can be
determined. Therapeutic, agricultural chemical, industrial chemical
and other products which benefit from modulation of such RNA attend
this result.
[0414] The simultaneous screening of a combinatorial library of
molecules of mass 700-750, against two nucleic acid targets of the
same molecular weight but different sequence, is demonstrated by
the use of mass modifying tags. If both nucleic acids targets being
studied are 27-mer RNAs of mass 8927, then screening a library of
molecules of mass 700-750 could afford a bewildering jumble of
noncovalent complex ions in the mass spectrum of the mixture of the
two nucleic acids and the library. However if one of the two
targets is mass modified, for example by the use of a PEG chain of
mass 3575 attached at the 5' terminus of the target, then the mass
spectrum will be significantly simplified. It is known that a
27-mer will generate multiply-charged ion signals, following
electrospray ionization, of mass/charge values 1486.8, 1784.4, and
2230.8 for the (M-6H).sup.6-, (M-5H).sup.5-, and the (M-4H).sup.4-
charge states. Upon binding to small molecules of mass 700-750, the
unmodified RNA-ligand complexes are anticipated to occur in the
1603.2-1611.6, 1924.4-1934.4, and 2405.8-2418.3 m/z range. If the
second nucleic acid target were not modified in any way, the
signals from its complexes would have occurred in the same regions.
However, using the mass modified RNA, bearing the PEG chain of mass
3575, results in the observation of the corresponding mass modified
RNA-ligand complexes to occur in the 2199-2207.4, 2639-2649 and
3299-3311 m/z range. Thus all signals from the second mass modified
nucleic acid would be cleanly resolved from those of the first RNA.
These noncovalent complex ions may be selected e.g. by triple,
quadrupole, ion trap or ICR techniques, and studied further by
MS/MS to afford detailed understanding of the sites of ligand-RNA
interaction, and the nature of these interactions, as has been
discussed above.
[0415] In a further embodiment, the methods of this invention are
applicable for the determination of the specificity of binding
interactions between a ligand and a biomolecular target. By
simultaneously screening multiple biomolecular targets with one or
more compounds, using the methods of this invention, it is possible
to ascertain whether a ligand binds specifically to only one target
biomolecule, or whether the binding observed with the target is
reproduced with control biomolecules as well, and is therefore
non-specific. This is an important distinction to be made when
screening large libraries and collections of compounds for binding
to biomolecular targets. It is desirable to quickly distinguish
those ligands that are selective or specific for the biomolecular
target of interest from those that are non-specific and bind to any
and all targets. From the standpoint of drug discovery, it is most
often the case that undesirable biological activities arise from
the indiscriminate, non-specific binding of molecules to unrelated
biomolecules. The present invention provides a valuable and
straightforward method for assessing the specificity of
interactions between a ligand and a panel of targets.
[0416] The use of mass modifying tags for the simultaneous
screening of multiple biomolecular targets is applicable to the
determination of binding specificity of a ligand as well. Mass
modifying tags may be used to differentiate several biomolecular
targets that serve as a control panel for screening a combinatorial
library of individual compounds against a specific biomolecular
target. When simultaneously screening multiple biomolecular targets
using the mass spectrometric methods of this invention, it is
necessary to ensure good separation of the ions that result from
each target and its complex with the binding ligand. This peak
overlap is easily eliminated by the facile introduction of
different mass modifying tags onto the different biomolecular
targets being studied. A mixture of the biomolecular target and the
control panel is mixed with the ligand being evaluated. This
solution is then ionized by ESI-MS, and the noncovalent complex
ions observed may be directly identified as having resulted from
the binding of the ligand to a specific target from the several
biomolecular targets present in the mixture. In this way, a
qualitative indication of specificity or selectivity of binding for
the desired target versus the control biomolecules may be obtained.
This selectivity may also be quantitated through the use of
appropriate standards of known binding affinity and comparison of
the ligand-biomolecule complex ion abundance to the abundance of
the standard-biomolecule abundance. Further, details on the nature
of the specific or non-specific interaction of the ligand with the
different biomolecules may also be obtained following ion-selection
and subsequent MS/MS experiments, as discussed above.
[0417] Likewise, it is also possible to determine the proportional
binding of a ligand to two or more biomolecular targets using the
methods of this invention. Thus by the use of appropriate mass
modifying tags on the different biomolecular targets, the different
noncovalent complexes formed via differential binding of the ligand
can be readily distinguished in the mass spectrometer. Quantitation
of the binding is possible by measuring the abundance of these
ions. Comparing the relative abundances of these ions provides a
means to determine the proportional binding of the ligand to the
different biomolecular targets.
[0418] Yet another application of the methods of the present
invention is to determine the differential binding of ligands to
biomolecular targets of different origin. When studying the binding
of small molecule ligands to RNA targets, it is straightforward to
distinguish between the noncovalent ligand-RNA complexes generated
from binding to the two different RNA targets, even though both may
be screened simultaneously as a mixture in the same assay. Further,
it is also possible to determine specificity and selectivity of the
ligand for one versus the other RNA, and to determine the relative
affinities of binding to each RNA target.
[0419] The methods of the present invention are applicable to the
study of a wide variety of biomolecular targets that include, but
are not limited to, peptides, proteins, receptors, antibodies,
oligonucleotides, RNA, DNA, RNA/DNA hybrids, nucleic acids,
modified oligonucleotides, peptide-nucleic acids (PNAs),
oligosaccharides, carbohydrates, and glycopeptides. Further these
biomolecular targets may be synthetic or isolated from natural
sources. Biomolecular targets of natural origin include, but are
not limited to, those obtained from microbial, plant, animal, viral
or human materials, such as, but not limited to, cells, cell
extracts, fluids, tissues and organs.
[0420] The molecules that may be screened by using the methods of
this invention include, but are not limited to, organic or
inorganic, small to large molecular weight individual compounds,
and combinatorial mixture or libraries of ligands, inhibitors,
agonists, antagonists, substrates, and biopolymers, such as
peptides or oligonucleotides.
[0421] Combinatorial mixtures include, but are not limited to,
collections of compounds, and libraries of compounds. These
mixtures may be generated via combinatorial synthesis of mixtures
or via admixture of individual compounds. Collections of compounds
include, but are not limited to, sets of individual compounds or
sets of mixtures or pools of compounds. These combinatorial
libraries may be obtained from synthetic or from natural sources
such as, for example to, microbial, plant, marine, viral and animal
materials. Combinatorial libraries include at least about twenty
compounds and as many as a thousands of individual compounds and
potentially even more. When combinatorial libraries are mixtures of
compounds these mixtures typically contain from 20 to 5000
compounds preferably from 50-1000, more preferably from 50-100.
Combinations of from 100-500 are useful as are mixtures having from
500-1000 individual species. Typically, members of combinatorial
libraries have molecular weight less than about 5000 Da.
[0422] The mass spectrometry techniques that may be used in the
methods of this invention include all of the techniques and systems
described herein or are subsequently developed. Tandem techniques
are also useful, including combinations of all of the foregoing and
LC/MS. The mass spectrometers used in the methods of this invention
may be a single quadrupole, triple quadrupole, magnetic sector,
quadrupole ion trap, time-of-flight instrument, and FTICR. Future
modifications to mass spectrometry are expected to give rise to
improved techniques which may also be useful herein.
[0423] The following examples are meant to be exemplary of
preferred embodiments of the invention and are not meant to be
limiting.
EXAMPLES
[0424] Determination of Molecular Interaction Sites
Example 1
The Iron Responsive Element
[0425] 1. Selecting RNA Target
[0426] To illustrate the strategy for identifying small molecule
interaction sites, the iron responsive element (IRE) in the mRNA
encoded by the human ferritin gene is identified. The IRE is a
typical example of an RNA structural element that is used to
control the level of translation of mRNAs associated with iron
metabolism. The structure of the IRE was recently determined using
NMR spectroscopy (#10372, #10504). In addition, NMR analysis of IRE
structure is described in Gdaniec, et al., Biochem., 1998, 37,
1505-1512 and Addess, et al., J. Mol. Biol., 1997, 274, 72-83. The
IRE is an RNA element of approximately 30 nucleotides that folds
into a hairpin structure and binds a specific protein. Because this
structure has been so well studied and it known to appear in the
mRNA of many speicies, it serves an an excellent example of how
Applicants' methodology works.
[0427] 2. Determining Nucleotide Sequence of the RNA Target
[0428] The human mRNA sequence for ferritin is used as the initial
mRNA of interest or master sequence. The ferritin protein sequence
is also used in the anayisis, particularly in the initial steps
used to find related sequences. In the case of human ferritin gene,
the best input is the full length annotated mRNA and protein
sequence obtained from UNIGENE. However, for many genes of interest
the same level of detailed information is not available. In these
cases, alternative sources of master sequence information is
obtained from sources such as, for example, GenBank, TIGR, dbEST
division of GenBank or from sequence information obtained from
private laboratories. Applicants' methods work using any level of
input sequence information, but reqTuires fewer steps with a high
quality annotated input sequence.
[0429] 3. Identifying Similar Sequences
[0430] An early step in the process is to use the master sequence
(nucleotide or protein) to find and rank related sequences in the
database (orthologs and paralogs). Sequence similarity search
algorithms are used for this purpose. All sequence similarity
algorithms calculate a quantitative measure of similarity for each
result compared with the master sequence. An expample of a
quantitative result is an E-value obtained from the Blast
algorithm. The E-values for a blast search of the non-redundant
GenBank database using ferritin mRNA as the query sesquence
illustrates the use of quantitative analysis of sequence similarity
searches. The E-value is the probability that a match between a
query sequence and a database sequence occurs due to random chance.
Therefore, the lower an E-value the more likely that two sequences
are truly related. A plot of the lowest E-value scores for ferritin
is shown in FIG. 10. Sequences that meet the cutoff criteria are
selected for more detailed comparisons according to a set of rules
described below. Since the objetive of the sequence similarity
search to find distantly related orthologs and paralogs it is
essential that the cutoff criteria not be too stringent, or the
target of the search will be excluded.
[0431] 4. Identification of Conserved Regions
[0432] Identification of conserved regions is performed by pairwise
sequence comparisons using Q-Compare in conjunction with
CompareOverWins. Conservation of structure between genes with
related function from different species is a major indication that
can be used to find good drug binding sites. Conserved structure
can be identified by using distantly related sequences and piecing
together the remnants of conseved sequence combining it with an
analysis of potential structure. Sequence comparisons are made
between pairs of mRNAs from different species using Q-compare that
can identify traces of sequence conservation from even very
divergent organisms. Q-compare, in conjuction with CompareOverWins,
compares every region of each sequence by sliding one sequence over
the other from end to end and measuring the number of matches in a
window of a specific size.
[0433] When the human mRNA and mouse mRNA sequences for ferritin,
which each contain an IRE in the 5'-UTR, are analyzed in this
manner, a plot showing the regions of sequence similarity is
produced, as shown in FIG. 5. Pairwise analysis of the human and
mouse ferritin mRNA sequences illustrate several important aspects
of this type of analysis. Regions of each mRNA that encode the
amino acid sequence have the highest degree of similarity, while
the untranslated regions are less similar. In FIG. 5, the location
of the IRE is indicated. In both the human and mouse ferritin mRNAs
the IREs are located in the extreme 5' end of each mRNA. This
demonstrates an important point--the sequence conservation in the
region of the IRE structure does not stand out against the
background of sequence similarity between the human and mouse
ferritin sequences. In contrast, in the comparison of human and
trout (FIG. 11) or human and chicken (FIG. 12) ferritin mRNAs, the
IREs can be immediately identified. This is because the sequence of
the UTRs between human and trout or human and chicken are separated
by greater evolutionarily distance than human and mouse, which is
logical in view of the evolutionary distance that separates humans
from birds and fish compared with other mammals. Comparing the
human sequence to that of birds and fish is informative because the
natural drift due to evolution has allowed many sequence changes in
the UTRs. However, the IRE sequences are more constrained because
they form an important structure. Thus, they stand out better and
can be more readily identified.
[0434] The same principle applies when comparing the trout and
chicken ferritin sequences to each other. While both are separated
from humans by hundreds of millions of years of evolution, they are
also well separated from each other. This illustrates another
important tactic used in the present invention--comparison of two
non-human RNA sequences can be used to find a regulatory RNA
structure without having the actual human sequence. The non-human
comparison work can actually direct one skilled in the art where to
look to find a human counterpart as a potential drug target.
[0435] Evolutionary distances can be used to decide which sequences
not to compare as well as which to compare. As with the human and
mouse, comparison of trout and salmon are less informative because
the species are too close and the IRE does not stand out above the
UTR background. Comparison of human and Drosophia ferritin mRNA
sequences fail to find the IREs in either species, even though they
are present. This is because the sequence of the IREs between
humans and Drosophila have diverged even though the structure is
conserved. However, if the Drosophila and mosquito ferritin mRNAs
are compared, the IREs are identified, again illustrating that the
human sequence need not be in hand to identify a regulatory element
relevant to drug discovery in humans.
[0436] The software used in the present invention makes the
decision whether or not to compare sequences pairwise using a
lookup table based upon the evolutionary distances between species.
An example of a small lookup table using the examples described
above is shown in FIG. 13. The lookup table in the present
invention includes all species that have sequences deposited in
GenBank. Q-Compare in conjunction with CompareOverWins decides
which sequences to compare pairwise.
[0437] 5. Identification Of Secondary Structure
[0438] Sets of sequences that show evidence of conservation in
orthologs and paralogs or other related genes are analyzed for the
ability to form internal structure. This is accomplished by
analyzing each sequence in a matrix where the seqeunce is plotted
5' to 3' on the X axis and its reverse complement is plotted 5' to
3' on the Y axis, such as in, for example, self-complementary
analysis. Matches that correspond to potential intramolecular base
pairs are scored according to a table of values. When the human
ferritin IRE sequence is analyzed in this fashion, the diagonals
indicate potential self-complementary regions. Each of the 13 IRE
sequences described in this example were analyzed in the same
fashion. While each of the sequences can form a variety of
different structures, the structure most likely to occur is one
common to all the sequences. By superimposing the plots of all 13
individual sequences (see, FIG. 8), the potential structure common
to all the sequences is deduced.
[0439] Synthesis of Ligands
Example 2
General Procedure for Automated Synthesis of Library Plates
[0440] ArgoGel-OHTM (360 mg, loading 0.43 mmole/g) was suspended in
.about.16 mL solution of 3:1 CH.sub.2Cl.sub.2/DMF. The suspension
was distributed equally among 12 wells of a 96 well polypropylene
synthesis plate (30 mg per well). The solvent was drained and the
resin dried overnight in vacuo over P.sub.2O.sub.5. All solid
reagents were dried in vacuo overnight over P.sub.2O.sub.5 prior to
use. For method 1, the Mitsunobu reagent 1 was dried, then
dissolved in anhydrous CH.sub.2Cl.sub.2 to a concentration of
0.15M. FMOC-Amino Acids (Novabiochem, Bachem Calif.) were dissolved
to a concentration of 0.30 M in a solution of 2:1 anhydrous
CH.sub.2Cl.sub.2/DMF for method 1, and to a concentration of 0.22 M
in DMF containing 0.44 M collidine for synthesis for method 2.
Sulfonyl chlorides were dissolved to a concentration of 0.2M in
Pyridine. Pyridine proved to be an acceptable solvent for most
sulfonyl chlorides, but when solubility was limited, cosolvents
such as MeCN, DMSO, CH.sub.2Cl.sub.2, DMF, and NMP (up to 50%) have
been employed. FMOC protection were removed with a solution of 10%
piperidine in anhydrous DMF prepared and used the day of synthesis.
Low water wash solvents were employed to ensure maximum coupling
efficiency of the initial amino-acid to the resin. Prior to loading
reagents, moisture sensitive reagent lines were purged with argon
for 20 minutes. Reagents were dissolved to appropriate
concentrations and installed on the synthesizer. Large bottles
(containing 8 delivery lines) were used for wash solvents and the
delivery of activator. Small septa bottles containing the amino
acids and sulfonyl chlorides allow anhydrous preparation and
efficient installation of multiple reagents by using needles to
pressurize the bottle, and as a delivery path. After all reagents
were installed, the lines were primed with reagent, flow rates
measured, then entered into the reagent table (.tab file) and the
dry resin loaded plate removed from vacuum and installed in the
machine for subsequent synthesis. After cleavage from support and
centrifugal evaporation of solvent, the products were dissolved in
MeOH/CH.sub.2Cl.sub.2 mixtures, then assayed for purity by TLC
(typically 10% MeOH/CH.sub.2Cl.sub.2) on silica gel using both UV
and 12 visualization, and for product identity by electrospray mass
spectroscopy (negative mode). Selected samples were dissolved in
DMSO-d.sub.6 and examined by .sup.1H NMR.
Example 3
General Hydroxamic Acid Synthesis Method 1 (FIG. 34)
[0441] The commercial ArgoGel-OH.TM. resin (10 .mu.mole) was washed
with CH.sub.2Cl.sub.2 (6.times.), then treated with the appropriate
FMOC-amino acid (3 eq.) and 1 (3 eq.). After 30 min, the wells were
drained, and the process repeated to give a total of 4 treatments
(12 eq.). The resin was washed with CH.sub.2Cl.sub.2 (6.times.),
DMF (4.times.), and the FMOC removed with 10% piperidine in DMF
(4.times.). The washes were collected, diluted appropriately, and
the amount of FMOC chromophore released quantitated by UV
(.di-elect cons.7800 L*mol.sup.-1*cm.sup.-1, .lambda.=301 nm). This
value was used to calculate the yield of the final products. The
resin was then washed with DMF (4.times.), then CH.sub.2Cl.sub.2
(6.times.), and treated with the appropriate sulfonyl chloride
(4.times.6 eq. for 15 min.) in pyridine, and washed with
CH.sub.2Cl.sub.2 (6.times.), DMF (6.times.), and CH.sub.2Cl.sub.2
(10.times.). At this point the resin could be treated with 90:5:5
TFA/H.sub.2O/Et.sub.3SiH for 4 h, then subjected to the above
washing procedure to remove any side chain protection on the
molecules if necessary. The plates were then removed from the
instrument, and individual wells treated with 4 M hydroxylamine
(50% aqueous) in 1,4-dioxane for 24 h. The filtrate was collected
into a deep well 96 well plate, the samples frozen, then
lyophilized to provide the desired hydroxamic acids. Addition of
fresh 1,4-dioxane and repetition of the lyophilization process
twice gave compounds free of any residual hydroxylamine (by .sup.1H
NMR of selected products).
Example 4
General Hydroxamic Acid Synthesis Method 2 (FIG. 35)
[0442] Resin 6 was prepared from ArgoGel-Wang-OH.TM. resin
according to publisched procedures and this resin (10 .mu.mole) was
washed with DMF (6.times.), CH.sub.2Cl.sub.2 (6.times.), then
treated with the appropriate FMOC-amino acid (3 eq.) in
DMF+collidine (6 eq.) and HATU (3 eq.). After 30 min, the wells
were drained, and the process repeated to give a total of 4
treatments (12 eq.). The resin was washed with CH.sub.2Cl.sub.2
(6.times.), DMF (4.times.), and the FMOC removed with 10%
piperidine in DMF (4.times.). The washes were collected, diluted
appropriately, and the amount of FMOC chromophore released
quantitated by UV (.di-elect cons.7800 L*mol.sup.-1*cm.sup.-1,
.lambda.=301 nm). This value was used to calculate the yield of the
final products. The resin was washed with DMF (4.times.), then
CH.sub.2Cl.sub.2 (6.times.), and treated with the appropriate
sulfonyl chloride (4.times.6 eq. for 15 min.) in pyridine, and
washed with CH.sub.2Cl.sub.2 (6.times.), DMF (8.times.), DMSO
(8.times.), and CH.sub.2Cl.sub.2 (10.times.). The plates were then
removed from the instrument, and individual wells treated with
90:5:5 TFA/Et.sub.3SiH/H.sub.2O for 4 h. The filtrate was collected
into a deep well 96 well plate, the resin washed (3.times.) with
TFA, and the samples concentrated in a centrifugal vacuum
concentrator. Addition of fresh 1,4-dioxane or isopropanol and
repetition of the concentration process twice, followed by drying
in vacuo overnight gave the desired hydroxamic acids.
[0443] The methods of both Examples 2 and 3 were utilized to
produce a library of compounds resulting from the combination of
FMOC-amino acids and sulfonyl chlorides shown in Table 2.
6TABLE 2 Reagents Used to Prepare Hydroxamic Acids 5 by Automated
Synthesis.sup.a FMOC-Amino Acid Used.sup.b Sulfonyl Chloride
Used.sup.c a D-Val.sup.d i 1-napthalene b D-Ile ii 2-napthalene c
D-Leu iii 2-thiophene d D-Ala iv 2-mesitylene e D-cyclo-hexyl-Ala v
3-nitrobenzene f D-norvaline vi 4-bromobenzene g D-norleucine vii
4-chlorobenzene h D-alloiso-leucine viii 4-iodobenzene i
D-.alpha.-t-Butylglycine.sup.e ix 4-nitrobenzene j D-Met x
4-methoxybenzene.sup.d k D-Phenyl-glycine xi 4-t-Butylbenzene l
D-Phe xii trifluoromethane.sup.d m D-4-Chloro-Phe xiii -toluene n
3-(2-napthyl)-D-Ala xiv 3-(trifluoromethyl)benzene o
3-(3-pyridyl)-D-Ala xv 4-(trifluromethoxy)benzene p
-(2-thienyl)-D-Ala xvi 4-(methylsulfonyl)benzene q D-Tyr(tBu).sup.d
xvii 4-(benzenesulfonyl)thiophene-2- r D-Trp xviii 4-ethylbenzene s
D-Cys(tBu) xix 4-cyanobenzene t S-Bn-D-penicillamine xx
4-methoxy-2,3,6-trimethylbenzene u glycine xxi
benzo-2,1,3-thiadiazole-4- v aminoisobutyric acid xxii
1-Methylimidazole-4- w D-Thr(tBu).sup.e xxiii
5-chloro-3-methylbenzo[B] thiophene-2-.sup.d x D-Ser(tBu) xxiv
benzofurazan-4- y D-His(Trt).sup.d xxv 3,5-dichlorobenzene z D-Pro
xxvi 3,4-dimethoxybenzene aa D-Tic xxvi 4-(n-butoxy)benzene i bb
D-Lys(BOC) xxvi 2,4-dichlorobenzene ii cc D-Asp(OtBu) xxix
4-trifluoromethylbenzene dd D-Glu(OtBu) xxx 2,5-dimethoxybenzene ee
L-Val xxxi 3,4-dichlorobenzene.sup.d ff L-Ala xxxi
4-n-propylbenzene.sup.d i gg L-Phe.sup.d xxxi
4-isopropylbenzene.sup.d ii hh D-Asn(Trt).sup.e xxxi
2,5-dichlorothiophene- iv 3- ii D-Gln(Trt).sup.e xxx 2-[1-methyl-5-
v (trifluoromethyl)pyrazol-3-yl] thiophene-5- jj D-Arg(Pmc).sup.d
xxx 2-[3- vi (trifluoromethyl)pyrid-2-yl sulfonyl]thiophene-5-
.sup.aAll possible combinations of reagents shown were utilized to
attempt the preparation of 1296 hydroxamic acids according to
method 2 (FIG. 35. .sup.bStandard abbreviations used for FMOC-amino
acids. All amino acids used were obtained from Novabiochem, Bachem,
or Synthetech. .sup.cTruncated chemical names are given in the
table. Appending `sulfonyl chloride` to the prefix listed gives the
appropriate name. All sulfonyl chlorides used were obtained from
Aldrich, Lancaster, or Maybridge. .sup.dAlso prepared via method 1
(FIG. 34). .sup.eFailed in method 1.
Example 5
Representative Parallel Array Synthesizer Input Files
[0444] The software inputs accept tab delimited text files from any
text editor. Examples for the synthesis of hydroxamic acids via the
procedure of FIG. 34 are shown in Table 3 (.cmd file), Table 4
(.seq file), and Table 5 (.tab file). Only several wells worth of
synthesis are shown for brevity. For an entire plate to be
prepared, only additional sulfonyl chlorides and additional amino
acids need to be added to the .tab file, and additional
combinations of the two need to be added to the seq file such that
it contains 96 lines, with each line corresponding to a unique
compound prepared.
[0445] The identity and purity of the compounds was determined by
electrospray mass spectroscopy (negative mode) and thin layer
chromatography on silica employing MeOH/CH.sub.2Cl.sub.2 solvent
mixtures (TLC). The synthesis products in approximately every third
well were assayed by TLC and electrospray mass spectroscopy, and
the desired compounds were generally present with purities of 60 to
90% when using either of the synthesis methods described above.
7TABLE 3 Example .cmd file (general synthesis procedure) which
executes the synthesis shown in FIG. 34. The cleavage from support
with hydroxy- lamine is performed separately. INITIAL_WASH BEGIN
Repeat 6 Add CH2Cl2 300 Drain 20 End_Repeat END COUPLE_AMINO_ACID
BEGIN Repeat 4 Add <SEQ> 100 + <ACT1> 200 Wait 1800
Drain 20 End_Repeat Repeat 6 Add CH2Cl2 300 Drain 20 End_Repeat
Repeat 4 Add DMF 300 Drain 20 End_Repeat END REMOVE_FMOC BEGIN
Load_Tray Repeat 4 Add PIPERIDINE_DMF 300 Wait 250 Drain 20
End_Repeat Remove_Tray Repeat 4 Add DMF 300 Drain 20 End_Repeat
Repeat 6 Add CH2Cl2 300 Drain 20 End_Repeat END
SULFONYLATE_AMINO_ACID BEGIN Next_Sequence Repeat 4 Add <SEQ>
300 Wait 900 Drain 20 End_Repeat Repeat 6 Add CH2Cl2 300 Drain 20
End_Repeat END FINAL_WASH BEGIN Repeat 6 Add DMF 300 Drain 20
End_Repeat Repeat 8 Add CH2Cl2 300 Drain 20 End_Repeat Repeat 2 Add
CH2Cl2 300 Drain 60 End_Repeat END
[0446]
8TABLE 4 Example .seq File (list of compounds to make) 1 A1 10
FMOC_D.sub.-- 4_MEO_BENZENE.sub.-- ALA SO2CL 2 A2 10 FMOC_D.sub.--
2_NAPTHYLENE.sub.-- VAL SO2CL 3 A3 10 FMOC_D.sub.--
3_CF3_BENZENE.sub.-- PHE SO2CL 4 A4 10 FMOC_D.sub.--
4_CL_BENZENE.sub.-- NAL SO2CL 5 A5 10 FMOC_D.sub.--
4_MEO_BENZENE.sub.-- SER(OTBU) SO2CL 6 A6 10 FMOC_D.sub.--
2_NAPTHYLENE.sub.-- ARG_PMC SO2CL 7 A7 10 FMOC_D.sub.--
3_CF3_BENZENE.sub.-- ALA SO2CL 8 A8 10 FMOC_D.sub.--
4_CL_BENZENE.sub.-- VAL SO2CL 9 A9 10 FMOC_D.sub.--
4_MEO_BENZENE.sub.-- PHE SO2CL 10 A10 10 FMOC_D.sub.--
2_NAPTHYLENE.sub.-- NAL SO2CL 11 A11 10 FMOC_D.sub.--
3_CF3_BENZENE.sub.-- SER(OTBU) SO2CL 12 A12 10 FMOC_D.sub.--
4_CL_BENZENE.sub.-- ARG_PMC SO2CL
[0447]
9TABLE 5 Example .tab (list of reagents to use) AMINO_ACIDS BEGIN 1
FMOC_D_ALA 265 0.30 2 FMOC_D_VAL 265 0.30 3 FMOC_D_PHE 265 0.30 4
FMOC_D_NAL 265 0.30 5 FMOC_D_SER(OTBU) 265 0.30 6 FMOC_D_ARG_PMC
265 0.30 END SOLVENTS BEGIN 67 CH2CL2 330 1 66 DMF 240 1 END
SULFONYLCHLORIDES BEGIN 9 4_MEO_BENZENE.sub.-- 220 0.20 SO2CL 10
2_NAPTHYLENE.sub.-- 220 0.20 SO2CL 11 3_CF3_BENZENE.sub.-- 220 0.20
SO2CL 12 4_CL_BENZENE.sub.-- 220 0.20 SO2CL END DEBLOCK BEGIN 68
PIPERIDINE_DMF 230 1 END ACTIVATORS BEGIN 69 BETAINE 300 0.15
Activates AMINO_ACIDS END
Example 6
Manual Solution Synthesis of Active Compounds
[0448] Methyl (2R)-2-amino-3-(2-naphthyl)propanoate.
[0449] To a suspension of D-napthylalanine hydrochloride (2.15 g,
10 mmole, Bachem Calif.) in MeOH (17 mL) was added TMS-C1 (2.8 mL,
22 mmole) dropwise with stirring. The mixture was allowed to stir
overnight, and the resulting solution concentrated in vacuo, then
dried over KOH to afford 2.65 g (100%) of methyl
(2R)-2-amino-3-(2-naphthyl)propanoate, which was >95% pure by
.sup.1H NMR, and used without further purification: R.sub.f0,63
(4:1:1 n-BuOH/AcOH/H.sub.2O); .sup.1H NMR (DMSO-d.sub.6) .delta.
8.76 (bs, 3H), 8.00-7.30 (m, 7H), 4.39 (t, 1H), 3.69 (s, 3H), 3.66
(m, 2H); MS (APCI.sup.+) m/e 230 (M+H).
[0450]
(2R)-2-(((4-bromophenyl)sulfonyl)amino)-3-(2-naphthyl)propanehydrox-
amic Acid (5-n-vi).
[0451] A suspension of D-Napthylalanine hydrochloride methyl ester
(1.33 g, 5 mmole), (i-Pr.sub.2)NEt (2.61 mL, 15 mmole) and
4-bromobenzesulfonyl chloride (1.53 g, 6 mmol) in CH.sub.2Cl.sub.2
(50 mL) was stirred at rt overnight. The solution was washed with
5% NaHCO.sub.3, dried (Na.sub.2SO.sub.4), concentrated, then
chromatographed (CH.sub.2Cl.sub.2 to 1% MeOH/CH.sub.2Cl.sub.2) and
concentrated to provide 2.05 g of the sulfonamide ester. This
material was dissolved in 1,4-dioxane (50 mL) and 25 mL of aqueous
hydroxylamine (50% w/w) was added. The mixture was allowed to stand
at rt for 48 h, then concentrated onto silica, chromatographed (2%
to 10% MeOH/CH.sub.2Cl.sub.2), the solid residue triturated with
water, and dried to provide 1.45 g (64%) of 5-n-vi: R.sub.f 0.35
(2% MeOH/CH.sub.2Cl.sub.2); .sup.1H NMR (DMSO-d.sub.6) .delta. 9.26
(bs, 1H), 7.90-7.20 (m, 11H), 3.88 (dd, 1H), 2.90 (m, 2H); MS
(electrospray.sup.-) m/e 447, 449 (M-H).
[0452] Anal. Calcd for
C.sub.19H.sub.17N.sub.2O.sub.4SBr.multidot.0.5 H.sub.2O: C, 49.79;
H, 3.96; N, 6.11. Found: C, 49.71; H, 3.90; N, 5.97.
[0453]
(2R)-3-(2-naphthyl)-2-((2-naphthylsulfonyl)amino)propanehydroxamic
Acid (5-n-ii).
[0454] A suspension of D-Napthylalanine hydrochloride methyl ester
(1.33 g, 5 mmole), (i-Pr.sub.2)NEt (2.61 mL, 15 mmole) and
4-napthalenesulfonyl chloride (1.36 g, 6 mmol) in CH.sub.2Cl.sub.2
(50 mL) was stirred at rt overnight. The solution was washed with
5% NaHCO.sub.3, dried (Na.sub.2SO.sub.4), concentrated, then
chromatographed (CH.sub.2Cl.sub.2 to 1% MeOH/CH.sub.2Cl.sub.2) and
concentrated to provide 2.02 g of the sulfonamide ester. This
material was dissolved in 1,4 -dioxane (50 mL) and 25 mL of aqueous
hydroxylamine (50% w/w) was added. The mixture was allowed to stand
at rt for 48 h, then concentrated onto silica, chromatographed (2%
to 10% MeOH/CH.sub.2Cl.sub.2), and dried to provide 1.15 g (55%) of
5-n-ii: R.sub.f 0.33 (2% MeOH/CH.sub.2Cl.sub.2); .sup.1H NMR
(DMSO-d.sub.6) .delta. 9.19 (bs, 2H), 8.17 (s, 1H), 7.95-7.35 (m,
12H), 7.17 (d, 1H), 3.97 (t, 1H), 2.83 (m, 2H); MS
(electrospray.sup.-) m/e 419 (M+H). Anal. Calcd for
C.sub.23H.sub.20N.sub.2O.sub.4S.multidot.0- .75 H.sub.2O: C, 63.85;
H, 4.99; N, 6.45. Found: C, 63.57; H, 4.74; N, 6.74.
Example 7
Antibacterial Testing
[0455] The crude compounds were screened in a representative high
throughput screening assay for antibacterial activity, and
compounds 5-n-ii and 5-n-vi were found to have activities minimum
inhibitory concentrations (MIC's) of 0.7-1.5 .mu.M and 3-6 .mu.M
against E. coli, respectively. This activity was verified by manual
solution synthesis of analytically pure material as described in
Example 6 above, which had identical activity.
Example 8
Functional Screening
[0456] The compounds are screened for binding affinity using MASS
or conventional high-throughput functional screens. The best
scoring compounds from docking a 256-member library against the 16S
A-site ribosomal RNA structure are shown in the table below. The
DOCK scores ranged from -308.8 to -144.2 as listed in Table 6. The
MASS assay was performed with the 27-mer model RNA sequence of the
16S A-site whose NMR structure has been determined. The
transcription/translation assay was based on expression of a
luciferase plasmid.
10TABLE 6 DOCK scores correlated with mass spectrometry and
biological assay DOCK MASS Compound score K.sub.D Activity.sup.1
Paromomycin -308.8 0.5 .mu.M 0.3 .mu.M 170046 -303.4 >50 >100
169999 -299.0 >50 >100 169963 -293.9 >50 >100 170070
-290.2 >50 >100 169970 -288.9 1.5 2.5 169961 -288.5 5.0 10
170003 -287.8 >50 >100 169995 -286.4 >50 >100 169993
-286.0 >50 >100 170072 -282.6 >50 >100 170078 -281.6
5.0 10 169985 -280.1 4.0 10 169998 -278.0 >50 >100
.sup.1Inhibition of protein synthesis in transcription/translation
assay for luciferase reporter.
[0457] Paromomycin is an aminoglycoside antibiotic known to bind to
the A-site RNA structure. The NMR structure was determined with
paromomycin bound at the A-site. Paromomycin had the best DOCK
contact score, along with high chemical and energy scores. The
docking results for these compounds have been correlated with their
binding affinity for 16S RNA fragment using MASS mass spectrometry,
and their ability to inhibit protein synthesis in a
transcription/translation assay. Four of the 12 compounds with the
best DOCK scores had good affinity (<10 .mu.M) for the RNA in
the MASS assay and inhibited translation of a luciferase plasmids
at <10 .mu.M. In addition, all 9 of the "good" binders in the
MASS assay scored in the top 30% in the DOCK calculation.
[0458] Ibis compound 169970 had the best energy score of any
compound, but a poor contact score. This result suggests that the
biological activity may be increased further by modifying the
structure to increase the number of close contacts with the 16S
A-site RNA.
Example 9
Target Site of TAR
[0459] The NMR solution structure of TAR RNA (Varani, et al., J.
Mol. Biol., 1995, 253, 313) has been used in the study of virtual
screening for HIV-1 TAR RNA ligands. The compounds present in the
Available Chemical Database (ACD) have been partitioned into a
number of subsets according to their formal charges (neutral, +1,
+2, etc) and DOCKed to the TAR structure. Five aminoglycoside
antibiotics were among the 20 compounds with the best binding
energies.
[0460] In addition, a number of compounds were docked to TAR with
subsequent evaluation of the solvation/desolvation energy. An
exemplary result is illustrated in FIG. 36 which shows that ACD
00001199 and ACD 00192509 show relatively low energies of
solvation/desolvation as well as low IC.sub.50 values.
Example 10
L11/Thiostrepton--An Example Of A High Throughput RNA/Protein
Assay
[0461] RNA molecules play a numerous roles in cellular functions
that range from structural to enzymatic in nature. These RNA
molecules may work as single large molecules, in complexes with one
or more proteins, or in partnership with one or more RNA molecules.
Some of these complexes, such as those found in the ribosome, have
been virtually intractable as high throughput screening targets due
to their immense size and complexity. The ribosome presents a
particularly rich source of RNA structures and functions that would
appear, at first glance, to be highly effective drug targets. A
large number of natural antibiotics exist that are directed against
ribosomal targets indicating the general success of this strategy.
These include the aminoglycosides, kirromycin, neomycin,
paromomycin, thiostrepton, and many others. Thiostrepton, a cyclic
peptide based antibiotic, inhibits several reactions at the
ribosomal GTPase center of the 50S ribosomal subunit. Evidence
exists that thiostrepton acts by binding to the 23S rRNA component
of the 50S subunit at the same site as the large ribosomal protein
L11. The binding of L11 to the 23S rRNA causes a large conformation
shift in the proteins tertiary structure. The binding of
thiostrepton to the rRNA appears to cause an increase in the
strength of the L11/23S rRNA interactions and prevents a
conformational transition event in the L11 protein thereby stalling
translation. Unfortunately, thiostrepton has very poor solubility,
relatively high toxicity, and is not generally useful as an
antibiotic. The discovery of new, novel, antibiotics directed
against these types of targets would be of great value.
[0462] The design of high throughput assays to discover new
antibiotics directed against ribosomal targets has been difficult,
in part, due to the large structures involved and the low binding
affinity of the RNA/protein interactions. Recently, a tremendous
amount of data has been generated concerning RNA structures in the
ribosome. This data has elucidated a number of structures and
enabled the prediction of many others. Further, the use of the SPA
assay format allows for assays to be run without washing or other
steps that lower the concentrations of binding components. This
allows one to examine binding interactions with very low (>1
.mu.M) Kd's.
[0463] The mode of action of thiostrepton appears to be to
stabilize a region of the 23S rRNA and by doing so prevent a
structural transition in the L11 protein. Among the many assays
that look at RNA/protein interactions, an SPA assay has been
designed to look for small molecules that could be effective as
thiostrepton `like` agents. This assay uses a radiolabeled small
fragment of the 23S rRNA, a biotinylated 75 amino acid fragment of
the L11 protein that contains the 23S rRNA binding domain and
thiostrepton. The folding conditions of the secondary and tertiary
structures of the 23S rRNA fragment have been examined as have the
binding conditions of the L11 fragment to the 23S rRNA. The
L11-thiostrepton assay has been optimized so that the 23S rRNA
fragment is in an unfolded state prior to the addition of
compounds. Addition of the L11 fragment to this unfolded RNA
results in no detectable binding interaction. The high throughput
assay is run by mixing the 23S rRNA fragment, under destabilizing
conditions, with compounds of interest, incubating this mixture,
and then adding the L11 fragment. Streptavidin-coated SPA beads are
added for binding detection. Thiostrepton is used as a positive
control. Addition of thiostrepton to the RNA promotes the correct
secondary and/or tertiary folding of the structure and allows the
L11 fragment to bind leading to the generation of a signal in the
assay.
[0464] A tested paradigm has been developed for designing,
developing and performing high and low throughput assays to look at
RNA/protein function, structure, and binding in bacteria. The
L11/thiostrepton assay described above is but one of a number of
RNA/protein interaction and functional assays that we have designed
and developed for high and low throughput screening. Others include
functional assays to measure RnaseP, RnaseE, and EF-Tu activity. An
assays to examine the function of the bacterial signal recognition
particle and S30 assembly is also contemplated.
Example 11
P48-4.5S Interaction
[0465] The P48 protein-binding region of the 4.5S RNA present in
the signal recognition particle of bacteria has been selected as a
target. The binding of P48 to 4.5S RNA is essential for bacteria to
survive, and development of an inhibitor of this binding should
generate a novel; class of antimicrobial agent. Using compounds
(.about.2.times.10.sup.5) from the Available Chemicals Directory
(ACD), as well as from additional libraries, initial screening
using DOCK (Meng, et al., J. Comp. Chem., 1992, 13, 505-524,
incorporated herein by reference in its entirety) (version 4.0) can
be carried out. This should leave about 15-20% of the database
which have reasonably good shape complementarity in docking to the
NMR structure of the 46mer, which is from the assymetric bulged
regions of E. coli 4.5S RNA. A pseudobrownian Monte Carlo search in
torsion angle space is performed using the program ICM (version
2.6), coupled with local minimization of each conformation, for
automated flexible docking of that truncated set of potential
ligands to the NMR structure and score for predicted affinity using
an empirical free energy function.
[0466] Approximately 2000 of the best scoring compounds will be
examined for experimental testing of their capability to inhibit
the binding of P48 to 4.5S RNA. Inhibition of P48-4.5S RNA binding
produced by the selected compounds will be measured using
(his).sub.6-tagged P48 and .sup.33P-RNA in a high-throughput
scintillation proximity assay system. The structure-activity
relationship among these 2000 compounds will serve as the basis for
an expanded synthetic effort.
[0467] Docking of small molecules to the region of the asymmetric
RNA bulges is expected to identify compounds with a high
probability of selectively destabilizing the 4.5S-P48 interaction
in vitro. The structure for the target RNA, shown in FIG. 37, will
be determined using NMR in the first phase of this proposal.
Compounds (approaching 2.times.10.sup.5) from the Available
Chemicals Directory (ACD) will be docked to the structure and
scored for predicted affinity. The best molecules will be screened
for their ability to disrupt the RNA-protein interaction.
Quantitative structure-activity relationship (QSAR) studies will be
performed on the most active compounds to identify critical
features and interactions with the RNA. New compounds
(.about.20,000) will be prepared through combinatorial addition
and/or repositioning of hydrogen bonding, aromatic, and charged
functional groups to enhance the activity and specificity of the
compounds for the bacterial SRP relative to the human counterpart.
In addition, a pseudobrownian Monte Carlo search in torsion angle
space using the program ICM2.6 (Abagyan, et al., J. Comp. Chem.,
1994, 15, 488-506, incorporated herein by reference in its
entirety) will be performed, coupled with local minimization of
each conformation, for automated flexible docking of the truncated
database to the NMR structural models.
[0468] In order to rank the ligands after flexible docking is
completed, a function to estimate their binding free energies is
used. There are a number of empirical methods for estimation of the
free energy of binding, but we intend to use the empirical free
energy function we derived from the thermodynamic binding cycle
(Filikov, et al., J. Comp.-Aided Molec. Design, 1998, 12, 1-12,
which is incorporated herein by reference in its entirety).
Example 12
Inhibition of Translation of an mRNA Containing a Molecular
Interaction Site by a "Small" Molecule Identified by Molecular
Docking
[0469] Translation of mRNAs in eukaryotic cells follows formation
of an initiation complex at the 5'-cap (m.sup.7Gppp). A variety of
initiation factors bind to the 5'-cap to form a pre-initiation
complex before the 40S ribosomal subunit binds to the
5'-untranslated region upstream of the AUG start codon. Pain, Eur.
J. Biochem., 1996, 236, 747-771. It has been demonstrated that RNA
secondary structures near the 5'-cap can affect the rates of
translation of mRNAs. Kozak, J. Biol. Chemistry, 1991, 266,
19867-19870. These RNA structures can bind proteins and inhibit the
level of translation. Standart, et al., Biochimie, 1994, 76,
867-879. The translational machinery has an ATP-dependant RNA
helicase activity associated with the eIF-4aleIF-4b complex, and
under normal conditions, the RNA structures are opened by the
helicase and do not slow the rate of translation of the mRNA. The
eIF-4a has a low (-.mu.M) affinity for the pre-initiation
complex.
[0470] It is believed that stabilization of mRNA structures near
the 5'-cap also could be effected by specific "small" molecules,
and that such binding would reduce the translational efficiency of
the mRNA. To test this hypothesis, a plasmid was constructed
containing the luciferase message behind a 5'-UTR containing a
27-mer RNA construct of the HIV TAR stem-loop bulge whose structure
had been determined by NMR. The resulting mRNA could be expressed
and capped in a wheat germ lysate translation system supplemented
with T7 polymerase following addition of m.sup.7G to the lysate
(see, FIG. 38A). Insertion of a 9-base leader before the TAR
structure (HIVluc+9) enhanced the translational efficiency,
presumably by allowing the pre-initiation complex to form. The
helicase activity associated with the pre-initiation complex can
transiently melt out the TAR RNA structure, and the message is
translated (see, FIG. 38A). Addition of a 39 amino acid tat peptide
to the lysate stabilized the TAR RNA structure and inhibited the
expression of the luciferase protein, as expected from a specific
interaction between the TAR RNA and tat (see, FIG. 38B).
[0471] "Small" organic molecules were then found that could inhibit
the translation of the TAR-luciferase mRNA by stabilizing the TAR
RNA structure. Compounds for the Available Chemicals Directory were
docked to the TAR RNA structure and scored for binding energies.
Among the best 25 compounds was ACD 00001199, whose structure is
shown below. This compound has been shown to bind to TAR RNA with
sufficient affinity to disrupt the interaction with tat peptide at
a 1 .mu.M concentration. 1
[0472] Addition of 00001199 to the wheat germ lysate translation
system with the luciferase mRNA produced some inhibition of
translation at very high concentrations (see, FIG. 39). However,
the compound was much more efficient in inhibiting translation of
the luciferase mRNA containing the TAR RNA structure in the 5'-UTR,
reducing translation by 50% at a 50 .mu.M concentrations. of small
molecules which do not bind specifically to the TAR RNA structure
did not affect translation of either mRNA construct (data not
shown).
Example 13
Determining the Structure of a 27-mer RNA Corresponding to the 16S
rRNA A Site
[0473] In order to study the structure of the 27-mer RNA
corresponding to the 16S rRNA A site, of sequence
5'-GGC-GUC-ACA-CCU-UCG-GGU-GAA-GUC-GCC-3- ' [SEQ. ID NO. 1] a
chimeric RNA/DNA molecule that incorporates three deoxyadenosine
(dA) residues at positions 7, 20 and 21 was prepared using standard
nucleic acid synthesis protocols on an automated synthesizer. This
chimeric nucleic acid of sequence
5'-GGC-GUC-dACA-CCU-UCG-GGU-GdAdA-- GUC-GCC-3' [SEQ. ID NO. 2] was
injected as a solution in water into an electrospray mass
spectrometer. Electrospray ionization of the chimeric afforded a
set of multiply charged ions from which the ion corresponding to
the (M-5H).sup.5- form of the nucleic acid was further studied by
subjecting it to collisionally induced dissociation (CID). The ion
was found to be cleaved by the CID to afford three fragments of m/z
1006.1, 1162.8 and 1066.2. These fragments correspond to the
w.sub.7.sup.(2-), w.sub.8.sup.(2-) and the a.sub.7-B.sup.(2-)
fragments respectively, that are formed by cleavage of the chimeric
nucleic acid adjacent to each of the incorporated dA residues.
[0474] The observation that cleavage and fragmentation of the
chimeric RNA/DNA has occurred adjacent to all three dA sites
indicates that the test RNA is not ordered around the locations
where the dA residues were incorporated. Therefore, the test RNA is
not structured at the 7, 20 and 21 positions.
[0475] A systematic series of chimeric RNA/DNA molecules is
synthesized such that a variety of molecules, each incorporating
deoxy residues at different site(s) in the RNA. All such RNA/DNA
members are comixed into one solution. MS analysis, as described
above, are conducted on the comixture to provide a complete map or
`footprint` that indicates the residues that are involved in
secondary or tertiary structure and those residues that are not
involved in any structure. See FIG. 40.
Example 14
Determining the Binding Site for Paromomycin on a 27-mer RNA
Corresponding to the 16S rRNA A Site
[0476] In order to study the binding of paromomycin to the RNA of
example 13, the chimeric RNA/DNA molecule of example 13 was
synthesized using standard automated nucleic acid synthesis
protocols on an automated synthesizer. A sample of this nucleic
acid was then subjected to ESI followed by CID in a mass
spectrometer to afford the fragmentation pattern indicating a lack
of structure at the sites of dA incorporation, as described in
Example 13. This indicated the accessibility of these dA sites in
the structure of the chimeric nucleic acid.
[0477] Next, another sample of the chimeric nucleic acid was
treated with a solution of paromomycin and the resulting mixture
analyzed by ESI followed by CID using a mass spectrometer. The
electrospray ionization was found to produce a set of multiply
charged ions that was different from that observed for the nucleic
acid alone. This was also indicative of binding of the paromomycin
to the chimeric nucleic acid, because of the increased mass of the
observed ion complex. Further, there was also observed, a shift in
the distribution of the multiply charged ion complexes which
reflected a change in the conformation of the nucleic acid in the
paromomycin-nucleic acid complex into a more compact structure.
[0478] Cleavage and fragmentation of the complex by CID afforded
information regarding the location of binding of the paromomycin to
the chimeric nucleic acid. CID was found to produce no
fragmentation at the dA sites in the nucleic acid. Thus paromomycin
must bind at or near all three dA residues. Paromomycin therefore
is believed to bind to the dA bulge in this RNA/DNA chimeric
target, and induces a conformational change that protects all three
dA residues from being cleaved during mass spectrometry. See FIG.
41.
Example 15
Determining the Identity of Members of a Combinatorial Library that
Bind to a Biomolecular Target
[0479] 1 .mu.L (0.6 O.D.) of a solution of a 27-mer RNA containing
3 dA residues (from Example 13) was diluted into 500 uL of 1:1
isopropanol:water and adjusted to provide a solution that was 150
mM in ammonium acetate, Ph 7.4 and wherein the RNA concentration
was 10 .mu.M. To this solution was added an aliquot of a solution
of paromomycin acetate to a concentration of 150 nM. This mixture
was then subjected to ESI-MS and the ionization of the nucleic acid
and its complex monitored in the mass spectrum. A peak
corresponding to the (M-5H).sup.5- ion of the paromomycin-27mer
complex is observed at an m/z value of 1907.6. As expected, excess
27-mer is also observed in the mass spectrum as its (M-5H).sup.5-
peak at about 1784. The mass spectrum confirms the formation of
only a 1:1 complex at 1907.6 (as would be expected from the
addition of the masses of the 27-mer and paromomycin) and the
absence of any bis complex that would be expected to appear at an
m/z of 2036.5.
[0480] To the mixture of the 27-mer RNA/DNA chimeric and
paromomycin was next added 0.7 .mu.L of a 10 .mu.M stock solution
of a combinatorial library such that the final concentration of
each member of the combinatorial library in this mixture with
27-mer target was 150 nM. This mixture of the 27-mer, paromomycin
and combinatorial compounds was next infused into an ESI-MS at a
rate of 5 .mu.L/min. and a total of 50 scans were summed (4
microscans each), with 2 minutes of signal averaging, to afford the
mass spectrum of the mixture.
[0481] The ESI mass spectrum so obtained, shown in FIG. 42,
demonstrated the presence of new signals for the (M-5H).sup.5- ions
at m/z values of 1897.8, 1891.3 and 1884.4. Comparing these new
signals to the ion peak for the 27-mer alone the observed values of
m/z of those members of the combinatorial library that are binding
to the target can be calculated. The masses of the binding members
of the library were determined to be 566.5, 534.5 and 482.5,
respectively. Knowing the structure of the scaffold, and
substituents used in the generation of this library, it was
possible to determine what substitution pattern (combination of
substituents) was present in the binding molecules.
[0482] It was determined that the species of m/z 482.5, 534.5 and
566.5 would be the library members that bore the acetic acid+MPAC
groups, the aromatic+piperidyl guanidine groups and the
MPAC+guanidylethylamide groups, respectively. In this manner, if
the composition of the combinatorial library is known a priori,
then the identity of the binding components is straightforward to
elucidate.
[0483] The use of FTMS instrumentation in such a procedure enhances
both the sensitivity and the accuracy of the method. With FTMS,
this method is able to significantly decrease the chemical noise
observed during the electrospray mass spectrometry of these
samples, thereby facilitating the detection of more binders that
may be much weaker in their binding affinity. Further, using FTMS,
the high resolution of the instrument provides accurate assessment
of the mass of binding components of the combinatorial library and
therefore direct determination of the identity of these components
if the structural make up of the library is known.
Example 16
Determining the Site of Binding for Members of a Combinatorial
Library that Bind to a Biomolecular Target
[0484] The mixture of 27-mer RNA/DNA chimeric nucleic acid, as
target, with paromomycin and the combinatorial library of compounds
from Example 15 was subjected to the same ESI-MS method as
described in Example 15. The ESI spectrum from Example 14 showed
new signals arising from the complexes formed from binding of
library members to the target, at m/z values of 1897.8, 1891.3 and
1884.4. The paromomycin-27mer complex ion was observed at an m/z of
1907.3.
[0485] Two complex ions were selected from this spectrum for
further resolution to determine the site of binding of their
component ligands on the 27-mer RNA/DNA chimeric. First, the ions
at 1907.3, that correspond to the paromomycin-27mer complex, were
isolated via an ion-isolation procedure and then subjected to CID.
No cleavage was found to occur and no fragmentation was observed in
the mass spectrum. This indicates that the paromomycin binds at or
near in the bulged region of this nucleic acid where the three dA
residues are present. Paromomycin therefore protects the dA
residues in the complex from fragmentation by CID.
[0486] Similarly, the ions at m/z 1897.8, that correspond to the
complex of a library member with the 27mer target, were isolated
via an ion-isolation procedure and then subjected to CID using the
same conditions used for the previous complex, and the data was
averaged for 3 minutes. The resulting mass spectrum (FIG. 43)
revealed six major fragment ions at m/z values of 1005.8, 1065.6,
1162.8, 2341.1, 2406.3 and 2446.0. The three fragments at m/z
1005.8, 1065.6 and 1162.8 correspond to the w.sub.6.sup.(2-),
a.sub.7B.sup.(2-) and w.sub.7.sup.(2-) ions from the nucleic acid
target. The three ions at higher masses of 2341.1, 2406.3 and
2446.0 correspond to the a.sub.20-B.sup.(3-) ion+566 Da,
w.sub.21.sup.(3-) ion+566 Da and the a.sub.21-B.sup.(3-) ion+566
Da. The data demonstrates at least two findings: first, since only
the nucleic acid can be activated to give fragment ions in this
ESI-CID experiment, the observation of new fragment ions indicates
that the 1897.8 ion peak results from a library member bound to the
nucleic acid target. Second, the library member has a molecular
weight of 566. This library member binds to the GCUU tetraloop or
the four base pairs in the stem structure of the nucleic acid
target (the RNA/DNA chimeric corresponding to the 16S rRNA A site)
and it does not bind to the bulged A site or the 6-base pair stem
that contains the U*U mismatch pair of the nucleic acid target.
[0487] Further detail on the binding site of the library member can
be gained by studying its interaction with and influence on
fragmentation of target nucleic acid molecules where the positions
of deoxynucleotide incorporation are different.
Example 17
Determining the Identity of a Member of a Combinatorial Library
that Binds to a Biomolecular Target and the Location of Binding to
the Target
[0488] A 10 .mu.M solution of the 27-mer RNA target, corresponding
to the 16S rRNA A-site that contains 3 dA residues (from Example
13), in 100 mM ammonium acetate at pH 7.4 was treated with a
solution of paromomycin acetate and an aliquot of a DMSO solution
of a second combinatorial library to be screened. The amount of
paromomycin added was adjusted to afford a final concentration of
150 nM. Likewise, the amount of DMSO solution of the library that
was added was adjusted so that the final concentration of each of
the 216 member components of the library was .about.150 nM. The
solution was infused into a Finnigan LCQ ion trap mass spectrometer
and ionized by electrospray. A range of 1000-3000 m/z was scanned
for ions of the nucleic acid target and its complexes generated
from binding with paromomycin and members of the combinatorial
library. Typically 200 scans were averaged for 5 minutes. The ions
from the nucleic acid target were observed at m/z 1784.4 for the
(M-5H).sup.5- ion and 2230.8 for the (M-4H).sup.4- ion. The
paromomycin-nucleic acid complex was also observed as signals of
the (M-5H).sup.5- ion at m/z 1907.1 and the (M-4H).sup.4 ion at m/z
2384.4 u.
[0489] Analysis of the spectrum for complexes of members of the
combinatorial library and the nucleic acid target revealed several
new signals that arise from the noncovalent binding of members of
the library with the nucleic acid target. At least six signals for
such noncovalent complexes were observed in the mass spectrum. Of
these the signal at the lowest m/z value was found to be a very
strong binder to the nucleic acid target. Comparison of the
abundance of this ligand-nucleic acid complex ion with the
abundance of the ion derived from the paromomycin-nucleic acid
complex revealed a relative binding affinity (apparent K.sub.D)
that was similar to that for paromomycin.
[0490] MS/MS experiments, with 6 minutes of signal averaging, were
also performed on this complex to further establish the molecular
weight of the bound ligand. A mass of 730.0.+-.2 Da was determined,
since the instrument performance was accurate only to .+-.1.5 Da.
Based on this observed mass of the bound ligand and the known
structures of the scaffold and substituents used in generating the
combinatorial library, the structure of the ligand was determined
to bear either of three possible combinations of substituents on
the PAP5 scaffold. The MS/MS analysis of this complex also revealed
weak protection of the dA residues of the hybrid RNA/DNA from CID
cleavage. Observation of fragments with mass increases of 730 Da
showed that the molecule binds to the upper stem-loop region of the
rRNA target.
Example 18
Determining the Identity of Members of a Combinatorial Library that
Bind to a Biomolecular Target and the Location of Binding to the
Target
[0491] A 10 .mu.M solution of the 27-mer RNA target, corresponding
to the 16S rRNA A-site that contains 3 dA residues (from Example
13), in 100 mM ammonium acetate at pH 7.4 was treated with a
solution of paromomycin acetate and an aliquot of a DMSO solution
of a third combinatorial library to be screened. The amount of
paromomycin added was adjusted to afford a final concentration of
150 nM. Likewise, the amount of DMSO solution of the library that
was added was adjusted so that the final concentration of each of
the 216 member components of the library was .about.150 nM. The
solution was infused into a Finnigan LCQ ion trap mass spectrometer
and ionized by electrospray. A range of 1000-3000 .mu.m/z was
scanned for ions of the nucleic acid target and its complexes
generated from binding with paromomycin and members of the
combinatorial library. Typically 200 scans were averaged for 5
minutes. The ions from the nucleic acid target were observed at m/z
1784.4 for the (M-5H).sup.5- ion and 2230.8 for the (M-4H).sup.4-
ion. The paromomycin-nucleic acid complex was also observed as
signals of the (M-5H).sup.5- ion at m/z 1907.1 and the
(M-4H).sup.4- ion at m/z 2384.4 u.
[0492] Analysis of the spectrum for complexes of members of the
combinatorial library and the nucleic acid target revealed several
new signals that arise from the noncovalent binding of members of
the library with the nucleic acid target. At least two major
signals for such noncbvalent complexes were observed in the mass
spectrum. MS/MS experiments, with .about.6 minutes of signal
averaging, were also performed on these two complexes to further
establish the molecular weights of the bound ligands.
[0493] The first complex was found to arise from the binding of a
molecule of mass 720.2.+-.2 Da to the target. Two possible
structures were deduced for this member of the combinatorial
library based on the structure of the scaffold and substituents
used to build the library. These include a structure of mass 720.4
and a structure of mass 721.1. MS/MS experiments on this
ligand-target complex ion using CID demonstrated strong protection
of the A residues in the bulge structure of the target. Therefore
this ligand must bind strongly to the bulged dA residues of the
RNA/DNA target.
[0494] The second major complex observed from the screening of this
library was found to arise from the binding of a molecule of mass
665.2.+-.2 Da to the target. Two possible structures were deduced
for this member of the library based on the structure of the
scaffold and substituents used to build the library. MS/MS
experiments on this ligand-target complex ion using CID
demonstrated strong fragmentation of the target. Therefore this
ligand must not bind strongly to the bulged dA residues of the
RNA/DNA target. Instead the fragmentation pattern, together with
the observation of added mass bound to fragments from the loop
portion of the target, suggest See FIG. 45. that this ligand must
bind to residues in the loop region of the RNA/DNA target.
Example 19
Simultaneous Screening of a Combinatorial Library of Compounds
Against Two Nucleic Acid Targets
[0495] The two RNA targets to be screened are synthesized using
automated nucleic acid synthesizers. The first target (A) is the
27-mer RNA corresponding to the 16S rRNA A site and contains 3 dA
residues, as in Example 13. The second target (B) is the 27-mer RNA
bearing 3 dA residues, and is of identical base composition but
completely scrambled sequence compared to target (A). Target (B) is
modified in the last step of automated synthesis by the addition of
a mass modifying tag, a polyethylene glycol (PEG) phosphoramidite
to its 5'-terminus. This results in a mass increment of 3575 in
target (B), which bears a mass modifying tag, compared to target
(A).
[0496] A solution containing 10 .mu.M target (A) and 10 .mu.M mass
modified target (B) is prepared by dissolving appropriate amounts
of both targets into 100 mM ammonium acetate at pH 7.4. This
solution is treated with a solution of paromomycin acetate and an
aliquot of a DMSO solution of the combinatorial library to be
screened. The amount of paromomycin added is adjusted to afford a
final concentration of 150 nM. Likewise, the amount of DMSO
solution of the library that is added is adjusted so that the final
concentration of each of the 216 member components of the library
is .about.150 nM. The library members are molecules with masses in
the 700-750 Da range. The solution is infused into a Finnigan LCQ
ion trap mass spectrometer and ionized by electrospray. A range of
1000-3000 m/z is scanned for ions of the nucleic acid target and
its complexes generated from binding with paromomycin and members
of the combinatorial library. Typically 200 scans are averaged for
5 minutes.
[0497] The ions from the nucleic acid target (A) are observed at
m/z 1486.8 for the (M-6H).sup.6- ion, 1784.4 for the (M-5H).sup.5-
ion and 2230.8 for the (M-4H).sup.4- ion. Signals from complexes of
target (A) with members of the library are expected to occur with
m/z values in the 1603.2-1611.6, 1924.4-1934.4 and 2405.8-2418.3
ranges.
[0498] Signals from complexes of the nucleic acid target (B), that
bears a mass modifying PEG tag, with members of the combinatorial
library are observed with m/z values in the 2199-2207.4, 2639-2649
and 3299-3311 ranges. Therefore, the signals of noncovalent
complexes with target (B) are cleanly resolved from the signals of
complexes arising from the first target (A). New signals observed
in the mass spectrum are therefore readily assigned as arising from
binding of a library member to either target (A) or target (B).
[0499] Extension of this mass modifying technique to larger numbers
of targets via the use of unique, high molecular weight neutral and
cationic polymers allows for the simultaneous screening of more
than two targets against individual compounds or combinatorial
libraries.
Example 20
Simultaneous Screening of a Combinatorial Library of Compounds
Against Two Peptide Targets
[0500] The two peptide targets to be screened are synthesized using
automated peptide synthesizers. The first target (A) is a 27-mer
polypeptide of known sequence. The second target (B) is also a
27-mer polypeptide that is of identical amino acid composition but
completely scrambled sequence compared to target (A). Target (B) is
modified in the last step of automated synthesis by the addition of
a mass modifying tag, a polyethylene glycol (PEG) chloroformate to
its amino terminus. This results in a mass increment of .about.3600
in target (B), which bears a mass modifying tag, compared to target
(A).
[0501] A solution containing 10 .mu.M target (A) and 10 .mu.M mass
modified target (B) is prepared by dissolving appropriate amounts
of both targets into 100 mM ammonium acetate at pH 7.4. This
solution is treated an aliquot of a DMSO solution of the
combinatorial library to be screened. The amount of DMSO solution
of the library that is added is adjusted so that the final
concentration of each of the 216 member components of the library
is .about.150 nM. The library members are molecules with masses in
the 700-750 Da range. The solution is infused into a Finnigan LCQ
ion trap mass spectrometer and ionized by electrospray. A range of
1000-3000 m/z is scanned for ions of the polypeptide target and its
complexes generated from binding with members of the combinatorial
library. Typically 200 scans are averaged for 5 minutes.
[0502] The ions from the polypeptide target (A) and complexes of
target (A) with members of the library are expected to occur at
much lower m/z values that the signals from the polypeptide target
(B), that bears a mass modifying PEG tag, and its complexes with
members of the combinatorial library Therefore, the signals of
noncovalent complexes with target (B) are cleanly resolved from the
signals of complexes arising from the first target (A). New signals
observed in the mass spectrum are therefore readily assigned as
arising from binding of a library member to either target (A) or
target (B). In this fashion, two or more peptide targets may be
readily screened for binding against an individual compound or
combinatorial library.
Example 21
Gas-Phase Dissociation of Nucleic Acids for Determination of
Structure
[0503] Nucleic acid duplexes can be transferred from solution to
the gas phase as intact duplexes using electrospray ionization and
detected using a Fourier transform, ion trap, quadrupole,
time-of-flight, or magnetic sector mass spectrometer. The ions
corresponding to a single charge state of the duplex can be
isolated via resonance ejection, off-resonance excitation or
similar methods known to those familiar in the art of mass
spectrometry. Once isolated, these ions can be activated
energetically via blackbody irradiation, infrared multiphoton
dissociation, or collisional activation. This activation leads to
dissociation of glycosidic bonds and the phosphate backbone,
producing two series of fragment ions, called the w-series (having
an intact 3'-terminus and a 5'-phosphate following internal
cleavage) and the a-Base series (having an intact 5'-terminus and a
3'-furan). These product ions can be identified by measurement of
their mass/charge ratio in an MS/MS experiment.
[0504] An example of the power of this method is presented in FIGS.
47 and 48. Shown in FIG. 47 part A is a graphical representation of
the abundances of the w and a-Base ions resulting from collisional
activation of the (M-5H).sup.5- ions from a DNA:DNA duplex
containing a G-G mismatch base pair. The w series ions are
highlighted in black and point toward the duplex, while the a-Base
series ions are highlighted in gray and point away from the duplex.
The more abundant the fragment ion, the longer and thicker the
respective arrow. Substantial fragmentation is observed in both
strands adjacent to the mismatched base pair. The results obtained
following collisional activation of the control DNA:DNA duplex ion
is shown in FIG. 47 part B. Some product ions are common, but the
pattern of fragmentation differs significantly from the duplex
containing the mismatched base pair. Analysis of the fragment ions
and the pattern of fragmentation allows the location of the
mismatched base pair to be identified unambiguously. In addition,
the results suggest that the gas phase structure of the duplex DNA
ion is altered by the presence of the mismatched pair in a way
which facilitates fragmentation following activation.
[0505] A second series of experiments with three DNA:RNA duplexes
are presented in FIG. 48. In the upper figure, an A-C mismatched
pair has been incorporated into the duplex. Extensive fragmentation
producing w and a-Base ions is observed adjacent to the mismatched
pair. However, the increased strength of the glycosidic bond in RNA
limits the fragmentation of the RNA strand. Hence, the
fragmentation is focussed onto the DNA strand. In the central
figure, a C-C mismatched base pair has been incorporated into the
duplex, and enhanced fragmentation is observed at the site of the
mismatched pair. As above, fragmentation of the RNA strand is
reduced relative to the DNA strand. The lower figure contains the
fragmentation observed for the control RNA:DNA duplex containing
all complementary base pairs. A common fragmentation pattern is
observed between the G5-T4 bases in all three cases. However, the
extent of fragmentation is reduced in the complementary duplexes
relative to the duplexes containing base pair mismatches.
Example 22
MASS Analysis of RNA--Ligand Complex to Determine Binding of Ligand
to Molecular Interaction Site
[0506] The ability to discern through mass spectroscopy whether or
not a proposed ligand binds to a molecular interaction site of an
RNA can be shown. FIGS. 49 and 50 depict the mass spectroscopy of
an RNA segment having a stem-loop structure with a ligand,
schematically illustrated by an unknown, functionalized molecule.
The ligand is combined with the RNA fragment under conditions
selected to facilitate binding and the result in complex is
analyzed by a multi target affinity/specificity screening (MASS)
protocol. This preferably employs electrospray ionization Fourier
transform ion cyclotron resonance mass spectrometry as described
hereinbefore and in the references cited herein. "Mass
chromatography" as described above permits one to focus upon one
bimolecular complex and to study the fragmentation of that one
complex into characteristic ions. The situs of binding of ligand to
RNA can, thus, be determined through the assessment of such
fragments; the presence of fragments corresponding to molecular
interaction site and ligand indicating the binding of that ligand
to that molecular interaction site.
[0507] FIG. 49 depicts a MASS Analysis of a Binding Location for a
non-A Site Binding molecule. The isolation through "mass
chromatography" and subsequent dissociation of the (M-5H)5- complex
is observed at m/z 1919.8. The mass shift observed in select
fragments relative to the fragmentation observed for the free RNA
provides information about where the ligand is bound. The (2-)
fragments observed below m/z 1200 correspond to the stem structure
of the RNA; these fragments are not mass shifted upon Complexation.
This is consistent with the ligand not binding to the stem
structure.
[0508] FIG. 50 shows a MASS Analysis of Binding Location for the
non-A Site Binding molecule. Isolation (i.e. "mass chromatography")
and subsequent dissociation of the (M-5H)5- complex observed at m/z
1929.4 provides significant protection from fragmentation in the
vicinity of the A-site. This is evidenced by the reduced abundance
of the w and a-base fragment ions in the 2300-2500 m/z range. The
mass shift observed in select fragments relative to the
fragmentation observed for the free RNA provides information about
where the ligand is bound. The exact molecular mass of the RNA can
act as an internal or intrinsic mass label for identification of
molecules bound to the RNA. The (2-) fragments observed below m/z
1200 correspond to the stem structure of the RNA. These fragments
are not mass shifted upon Complexation--consistent with ligand not
being bound to the stem structure. Accordingly, the location of
binding of ligands to the RNA can be determined.
Example 23
Determination of Specificity and Affinity of Ligand Libraries to
RNA Targets
[0509] A preferred first step of MASS screening involves mixing the
RNA target (or targets) with a combinatorial library of ligands
designed to bind to a specific site on the target molecule(s).
Specific noncovalent complexes formed in solution between the
target(s) and any library members are transferred into the gas
phase and ionized by ESI. As described herein, from the measured
mass difference between the complex and the free target, the
identity of the binding ligand can be determined. The dissociation
constant of the complex can be determined in two ways: if a ligand
with a known binding affinity for the target is available, a
relative Kd can be measured by using the known ligand as an
internal control and measuring the abundance of the unknown complex
to the abundance of the control, alternatively, if no internal
control is available, Kd's can be determined by making a series of
measurements at different ligand concentrations and deriving a Kd
value from the "titration" curve.
[0510] Because screening preferably employs large numbers of
similar, preferably combinatorially derived, compounds, it is
preferred that in addition to determining whether something from
the library binds the target, it is also determined which
compound(s) are the ones which bind to the target. With highly
precise mass measurements, the mass identity of an unknown ligand
can be constrained to a unique elemental composition. This unique
mass is referred to as the compound's "intrinsic mass label." For
example, while there are a large number of elemental compositions
which result in a molecular weight of approximately 615 Da, there
is only one elemental composition (C.sub.23H.sub.45N.sub.5O.sub.14)
consistent with a monoisotopic molecular weight of 615.2963012 Da.
For example, the mass of a ligand (paromomycin in this example)
which is noncovalently bound to the 16S A-site was determined to be
615.2969+0.0006 (mass measurement error of 1 ppm) using the free
RNA as an internal mass standard. A mass measurement error of 100
ppm does not allow unambiguous compound assignment and is
consistent with nearly 400 elemental compositions containing only
atoms of C, H, N, and O. The isotopic distributions shown in the
expanded views are primarily a result of the natural incorporation
of 13C atoms; because high performance FTICR can easily resolve the
12C-13C mass difference we can use each component of the isotopic
cluster as an internal mass standard. Additionally, as the
theoretical isotope distribution of the free RNA can be accurately
simulated, mass differences can be measured between "homoisotopic"
species (in this example the mass difference is measured between
species containing four 13C atoms).
[0511] Once the identity of a binding ligand is determined, the
complex is isolated in the gas phase (i.e. "mass chromatography")
and dissociated. By comparing the fragmentation patterns of the
free target to that of the target complexed with a ligand, the
ligand binding site can be determined. Dissociation of the complex
is performed either by collisional activated dissociation (CAD) in
which fragmentation is effected by high energy collisions with
neutrals, or infrared multiphoton dissociation (IRMPD) in which
photons from a high power IR laser cause fragmentation of the
complex.
[0512] A 27-mer RNA containing the A-site of the 16S rRNA was
chosen as a target for validation experiments. See FIG. 51. The
aminoglycoside paromomycin is known to bind to the unpaired
adenosine residues with a Kd of 200 nm and was used as an internal
standard. The target was at an initial concentration of 10 mM while
the paromomycin and each of the 216 library members were at an
initial concentration of 150 nm. While this example was performed
on a quadrupole ion trap which does not afford the high resolution
or mass accuracy of the FTICR, it serves to illustrate the MASS
concept. Molecular ions corresponding to the free RNA are observed
at m/z 1784.4 (M-5H+)5- and 2230.8 4 (M-4H+) 4-. The signals from
the RNA-paromomycin internal control are observed at m/z 1907.1 4
(M-5H+)5- and 2384.4 4 (M-4H+)4-. In addition to the expected
paromomycin complex, a number of complexes are observed
corresponding to binding of library members to the target. See FIG.
52.
[0513] One member of this library (MW=675.8+1.5) forms a strong
complex with the target but MS/MS studies reveal that the ligand
does not offer protection of A-site fragmentation and therefore
binds to the loop region. Another member of Isis 113069 having an
approximate mass of 743.8+1.5 demonstrates strong binding to the
target and, as evidenced by MS/MS experiments provides protection
of the unpaired A residues, consistent with binding at the
A-site.
[0514] The rapid and parallel nature of the MASS approach allows
large numbers of compounds to be screened against multiple targets
simultaneously, resulting in greatly enhanced sample throughput and
information content. In a single assay requiring less than 15
minutes, MASS can screen 10 targets against a library containing
over 500 components and report back which compounds bind to which
targets, where they bind, and with what binding affinity.
Sequence CWU 1
1
24 1 27 RNA Artificial Sequence Synthetic construct 1 ggcgucacac
cuucggguga agucgcc 27 2 27 RNA Artificial Sequence Synthetic
construct 2 ggcgucacac cuucggguga agucgcc 27 3 24 RNA Artificial
Sequence Synthetic construct 3 uaaggaggug auaucaccuc cuua 24 4 23
RNA Artificial Sequence Synthetic construct 4 cugcuucaac agugcuugga
cgg 23 5 23 RNA Artificial Sequence Synthetic construct 5
cugcuucaac agugcuugaa cgg 23 6 23 RNA Artificial Sequence Synthetic
construct 6 cugcgucaac agugcuugga cgg 23 7 23 RNA Artificial
Sequence Synthetic construct 7 uugcuucaac agugauugaa cgg 23 8 23
RNA Artificial Sequence Synthetic construct 8 uugcuucaac aguguuugaa
cgg 23 9 23 RNA Artificial Sequence Synthetic construct 9
cuucugcgcc agugugugua aag 23 10 23 RNA Artificial Sequence
Synthetic construct 10 cuucugugcc aguguguaua aag 23 11 46 RNA
Artificial Sequence Synthetic construct 11 ggacgcuacu cuguuuacca
gguucgccaa ggcagaugac gcgucc 46 12 27 RNA Artificial Sequence
Synthetic construct 12 ggcgucacac cuucggguga agucgcc 27 13 27 RNA
Artificial Sequence Synthetic construct 13 ggcgucacac cuucgggugu
agacgcc 27 14 12 RNA Artificial Sequence Synthetic construct 14
caccuucggg ug 12 15 14 DNA Artificial Sequence Synthetic construct
15 cgcttgggag tctc 14 16 14 DNA Artificial Sequence Synthetic
construct 16 gagactgcca agcg 14 17 14 DNA Artificial Sequence
Synthetic construct 17 cgcttggcag tctc 14 18 14 DNA Artificial
Sequence Synthetic construct 18 gagactgcca agcg 14 19 14 DNA
Artificial Sequence Synthetic construct 19 agcttagcag tctc 14 20 14
RNA Artificial Sequence Synthetic construct 20 gagacugcca agcu 14
21 14 DNA Artificial Sequence Synthetic construct 21 agcttgccag
tctc 14 22 14 RNA Artificial Sequence Synthetic construct 22
gagacugcca agcu 14 23 14 DNA Artificial Sequence Synthetic
construct 23 agcttggcag tctc 14 24 14 RNA Artificial Sequence
Synthetic construct 24 gagacugcca agcu 14
* * * * *
References